Rozprawy doktorskie na temat „Single Stranded DNA Binding Protein (SSBb)”

Single-stranded DNA binding proteins (SSBs) are critical for binding, protecting and sequestering single-stranded DNA intermediates during multiple cellular transactions, including DNA replication, repair and transcription. The canonical SSB in eukaryotes, Replication Protein A (RPA), is a heterotrimeric protein essential for numerous cellular processes, including DNA repair by homologous recombination (HR). Recently, Richard et al. (2008) identified a novel human SSB, designated human Single-Stranded DNA Binding protein 1 (hSSB1), critical to DNA repair and the maintenance of genomic stability. siRNA-mediated depletion of hSSB1 led to attenuation of ATM signalling in response to DNA damage by ionizing radiation (IR), impairment of DNA repair by HR, and overall genetic instability. Moreover, hSSB1 was subsequently shown to itself function in a heterotrimeric complex in a manner analogous to RPA, with Integrator complex subunit 3 (INTS3), and a small, uncharacterised acidic protein C9Orf80/MISE/SSBIP1. siRNA-mediated depletion of these components led to similar DNA damage-related phenotypes to what has been observed for hSSB1 depletion alone, suggesting that complex formation may be important for hSSB1 functioning. Moreover, hSSB2, a homolog of hSSB1, was shown to be able to form a similar complex with INTS3 and C9Orf80 in place of hSSB1, suggesting an element of functional redundancy in the roles of hSSB1 and hSSB2.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair. Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3' end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components. Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called bubble migration synthesis, a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Applied and Material Sciences." Discipline: Applied and Materials Sciences; Department/School: Applied and Materials Sciences.

Single stranded DNA (ssDNA) binding proteins (SSBPs), are known key players of DNA damage response (DDR) pathway and play an essential role in stabilising fragile ssDNA generated during DNA replication, transcription and repair. The canonical SSBP is the heterotrimeric Replication Protein A (RPA) which is involved in a number of key cellular processes including replication and repair via Homologous Recombination (HR) in the course of DNA damage. Our lab recently described two new SSBPs, termed SSB1 and SSB2 (also known as NABP2/OBFC2B/SOSS-B1 and NABP1/OBFC2A/SOSSB-2, respectively) which form independent co-complexes with two additional proteins, the Integrator complex subunit 3 (INTS3) and the chromosome 9 open reading frame 80 (C9ORF80), a small acidic 104 residue polypeptide. Previously, we demonstrated that whilst Ssb1/Nabp2 KO in mouse caused perinatal lethality, Ssb2/Nabp1 KO did not lead to any phenotypic abnormalities. Interestingly, ablation of Ssb1 led to stabilisation of Ssb2 and vice-versa, indicating functional redundancy between these two proteins. This was recently demonstrated in-vivo by the generation of Ssb1 and Ssb2 (together referred as Ssb1/2) double-knockout (DKO) mice, which caused early embryonic lethality in a constitutive model and acute bone marrow failure and intestinal atrophy using the inducible Rosa26-CreERT2 system. To delineate the functional redundancy between these two proteins at the molecular level, we have generated inducible DKO mouse embryonic fibroblasts (MEFs) using the Rosa26-CreERT2 system, which will be described in the first research chapter. We found that cumulative loss of Ssb1/2 in the primary as well as SV40-immortalised MEFs led to acute proliferation arrest and cell death following TAM administration. This was associated with accumulation of genomic instability via endogenous replication stress. Although loss of Ssb1/2 in-vivo and in-vitro is associated with accumulation of R-loops, the overall DKO phenotype was not able to be rescued with overexpression of RNaseH1, which resolves R-loops. Additionally, we investigated the roles of Ssb1/2 following treatment with different DNA damaging agents to determine their roles in the DDR system. Interestingly, DDR signalling in DKO was normal following ionizing radiation, ultraviolet C and camptothecin but with hydroxyurea treatment that causes replication stress, we observed a delayed signalling response in DKO. Together, this chapter defines the phenotypic changes that take place in-vitro when Ssb1 and Ssb2 are deleted. The second research chapter describes our finding that loss of Ssb1 and Ssb2 together leads to reduced levels of several Integrator components and thus, has an equivalent profound effect on the misprocessing of the Sm- associated small nuclear RNAs (snRNAs) to that of the Integrator catalytic components- IntS9 and IntS11. Here, we show that upregulated snRNAs are not only misprocessed, but extend up to several hundred to a thousand base pairs past their native termination site, and are polyadenylated. Additionally, we demonstrate that loss of Ssb1/2 led to changes in the dynamics of alternative splicing, likely due to perturbation of the splicing machinery by aberrant snRNAs. We further show that a number of regulators of transcription and the cell cycle are affected by these changes, which might contribute to the loss of viability observed in DKO cells. Together, these findings reveal the critical role of Ssb1/2 and their association with the Integrator complex in regulating cellular proliferation and spliceosomal function. The third chapter of this thesis further investigates the intestinal atrophy observed upon loss of Ssb1 and Ssb2 in the DKO mice from our lab. For this, we have generated a small intestine (SI) specific Ssb1/2 DKO mouse- the VillinCreERT2 Ssb1flox/flox; Ssb2flox/flox model. This mouse model is a unique system to study the undefined roles of Ssb1/2 in the small intestine (SI) by bypassing the confounding effects of the bone marrow phenotype in the ubiquitous Rosa26-CreERT2 DKO model. We have found that loss of intestinal Ssb1/2 leads to exhaustion of the stem cells in the crypts, resulting in loss of the normal crypt-villus axis anatomy which causes acute morbidity within six days of induction. Interestingly, the stem cells are pushed to proliferate immediately after the loss of Ssb1 and Ssb2, followed by the exhaustion of these cells. This is demonstrated by sequential proliferation studies using the known thymidine analogue 5-bromo-2’deoxyuridine (BrdU) as well as quantitative reverse transcription polymerase chain reaction (qRT-PCR). Therefore through this model, we have demonstrated a fundamental role of Ssb1/2 in the maintenance of intestinal homeostasis. In conclusion, through the inducible abrogation of two SSBPs- Ssb1 and Ssb2 together, we have demonstrated several novel roles of these proteins in the maintenance of genomic stability both in-vitro and in-vivo that were previously masked in single KO studies. Further, we have defined the molecular mechanisms underlying the acute lethality observed upon abrogation of these two proteins.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

The Incil plasmid Colib-P9 was found to carry a single-stranded DNA-binding protein gene (ssb), and the cloned gene was able to suppress the UV and temperature-sensitivity of an ssb-l strain of Escherichia coli K-12. Determination of the nucleotide sequence of Colib ssb demonstrated that the gene shows considerable homology to the ssb gene of plasmid F. In contrast, Southern hybridization techniques indicated that the IncP plasmid RP4 lacks a gene with any extensive homology to F ssb. It was shown that the direction of transfer of Colib-P9 is such that the Colib ssb gene, which lies approximately 11 kb from the origin of transfer, is located within the region transferred early during conjugation. The Colib and F ssb genes are therefore similarly located on their respective plasmids. The Colib ssb gene was shown to be coordinately expressed with the transfer (tra) genes, suggesting that the Colib SSB protein may participate in the conjugative process. However, a mutant Colibdrd-1 derivative carrying a Tn903-derived insertion in ssb showed no defect in tests of conjugative efficiency and was apparently maintained stably both following mating and during vegetative growth. Thus no biological role for the Colib SSB protein was detected. However, unlike the parental plasmid, the Colib ssb mutants conferred a marked Psi- (plasmid- mediated SOS inhibition) phenotype on recA441 and recA730 strains. This may result from high level expression of a psi gene due to readthrough from the Tn903 insertion. It is now apparent that many conjugative plasmids previously thought to be unrelated may be derived from a common ancestral plasmid which possessed both ssb and psi genes. It is speculated that the function of the SSB proteins of conjugative plasmids such as Colib and F may subsequently have been duplicated by analogues derived from newly aquired conjugation systems.

Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). The APs belong to the COG3390 group of proteins found in Euryarchaeota and feature an OB fold. Genetic analysis of deletion mutants was employed to determine if all three RPAs are essential for cell viability, and if there is an element of redundancy between RPA1 and RPA3. The hypothesis that the RPAs form a complex with their respective APs, as opposed to a heterotrimeric RPA complex, was also investigated. Furthermore, it was tested whether the RPAs and their respective APs are specific for each other, or whether they are interchangeable. The genetic analysis showed that RPA2 is essential for cell viability, but that neither RPA1 nor RPA3 are. The rpa3, rpa3ap and the rpa3 operon deletion mutants showed sensitivity to DNA damage but only a slight growth defect. By contrast, the rpa1, rpa1ap, rpe and rpa1 operon mutants did not show any DNA damage sensitivity and an even milder growth defect. The double rpa1 rpa3 operon deletion was difficult to generate but unexpectedly lacked a significant DNA damage sensitivity and growth defect. The inability to make the double rpa1 rpa3ap and rpa1ap rpa3 deletion mutants suggests that the APs are specific for their respective RPAs. Biochemical analysis involving histidine-tagged RPAs and APs was used to confirm the conclusions of the genetic analysis. The RPAs did not interact with each other, but instead co-purified with their respective APs. This finding reiterates that the RPAs do not form a heterotrimeric complex, as seen in eukaryotes, but instead form a novel complex with their respective APs.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

Human single-stranded DNA-binding protein 1 (hSSB1) is required for the timely repair of double-strand DNA breaks, as well as the stabilisation and restart of stalled replication forks. In this work, evidence is provided that cellular survival in response to replication stress is promoted by dynamic phosphorylation of hSSB1 by the DNA-dependent protein kinase (DNA-PK) and PPP-family protein phosphatases. These data provide insight into the functional regulation of hSSB1 following replication fork disruption.

Thesis (Ph. D.)--University of Toledo, 2008.
Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry." Includes bibliographical references (leaves 305-309).

Genetic instability is the driver of cancer initiation and progression. The human single stranded DNA binding protein 2 is known to be involved in the prevention of such genetic instability. This project has highlighted how human single stranded DNA binding protein 2 is involved in a particular DNA repair pathway called, base excision repair. The researcher identified the role of human single stranded DNA binding protein 2 in the removal of uracils that have been added by mistake to the human genome. This project details the mechanism by which uracils are removed from the genome, shedding light on the evolution of the cancer genome and the mechanism through which genetic stability can occur.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.

Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.

The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.

Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.

Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.

Keywords:apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

碩士
國立清華大學
生物科技研究所
93
Single strand DNA binding protein (SSB) plays essential roles in many processes related to DNA metabolism such as DNA replication, repair, and homologous genetic recombination. The HP1245 gene was annotated as SSB in Helicobacter pylori strain 26695. However, there are no functional or structured studies for this SSB up to now. The full length (179 residues), three different C-terminal truncated species (106, 122 and 134 residues) and four different site directed mutants (F37A, F50A, F56A and W84A) were individually sub-cloned into pQE30 vector and expressed in E. coli SG13009. After IPTG induction, each species of recombinant (rec) HP1245 protein with N-terminally 6xHis-tagged fusion was purified by Ni-NTA affinity chromatography and its identity confirmed by mass spectrometry and Western blotting analysis using an anti-His-tag monoclonal antibody. Full length and various mutant recHP1245 proteins were homo-tetramer according to mass spectrometry and sedimentation velocity ultracentrifuge analysis, respectively. However, even in NaCl containing buffer, full length recHP1245 protein was easy to degrade after stored at 4℃ for 2 weeks, but C-terminal truncated proteins (122 and 134 residues) were stable for months. This indicated that the inherently disordered C-terminal region of SSB in H. pylori, similar to that in E. coli, may affect protein stability. The ssDNA binding property of HP1245 was performed through electrophoretic mobility shift assays (EMSA) to determine the binding affinity and the binding domain. The result indicated that the affinity of full-length or truncated recHP1245 proteins (122 and 134 residues) with biotin-labeled d(T)35 ssDNA was similar. It meant that the ssDNA binding domain was located at the N terminal. Furthermore, the site directed mutants, F37A, F50A, F56A and W84A were also individually measured by the same method. These results indicated that either F56A or W84A residue of recHP1245 protein would decrease the ssDNA binding affinity. Therefore, the two residues played a crucial role on ssDNA binding. These results suggested that the aromatic residues in this protein might contribute certain roles on ssDNA binding via base stacking interaction with the base in the ssDNA. In order to survive in stomach, Helicobacter pylori must have the ability to modify gene expression in acidic circumstances. To investigate whether HP1245 protein could be acid-induced or not at this stress, H. pylori were individually cultured on Brucella agar plates for 48 h at pH 7.0 and pH 5.5. In this study, the result showed that the protein expression of HP1245 did not have significant difference.

Genomic integrity is a fundamental requisite for survival and proliferation of all organisms. The genetic material is continuously threatened by a multitude of extrinsic and intrinsic factors. Consequently, the presence of strong DNA repair systems is essential to aid errorfree transmission of genetic material to successive generations. In prokaryotes, repair by homologous recombination (HR) provides a major means to reinstate the genetic information lost in DNA damage. Pathogenic bacteria, such as Mycobacterium tuberculosis, face an additional threat of DNA damage due to antibiotic treatment and immune stresses inside the macrophage. Consequently, M. tuberculosis has evolved a remarkably strong DNA repair network, providing it robust survivability in the harsh environments faced inside the host cell. The importance of HR or recombination repair in pathogenesis and emergence of antibiotic resistance in M. tuberculosis is well established. However, many aspects of the pathway remain elusive as of now. This thesis is concerned with the analysis of recombination repair system in the genus mycobacteria and characterization of two novel proteins identified in the process. Chapter 1 gives a detailed account of the mechanics of HR, using the well studied E. coli system and highlights the differences in mycobacteria. Recombination repair comprises a series of processes carried out by more than 20 proteins, that ultimately leads to repair of damaged DNA. Processing of DNA strands at double strand breaks and single strand gaps, to produce a 3' overhang, initiates the process. Unlike E. coli, complexes of RecFO-RecR and AdnAB-RecR provide two alternate pathways for end resection of strands and RecA loading in mycobacteria. The exchange of an undamaged strand with the damaged strand, facilitated by RecA, is central to recombination. Additionally, Single-Stranded DNA binding proteins (SSB) facilitate the loading of RecA onto the single-stranded overhang produced by the pre-processing enzymes. Resolution of strands formed due to strand exchange, via multi-stand branched DNA intermediates (such as D-loops, three-way junctions, Holliday junctions etc) by RuvABC or RecG, is the final step of recombination. An additional HJ resolvase YqgF, with unclear functions, is also present in mycobacteria. Furthermore, the major end resection enzymes (RecBCD) involved in HR in E. coli, were implicated in the Single-Strand Annealing (SSA) pathway in mycobacteria. As part of an effort to improve the understanding of recombination repair in mycobacteria, a structure based genomic search for such proteins was carried out in 43 mycobacteria with known genome sequences (Chapter 2). Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the same protein from different strains suggests the importance of RecO-RecA and RecFORRecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New insights into the binding of small molecules to the relevant proteins are provided by binding pocket analysis using three-dimensional structural models. New annotations obtained from the analysis, include identification of a protein (RecGwed) with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. A second SingleStranded DNA Binding protein (SSBb), in addition to the canonical one (SSBa), was present in all mycobacteria except M. leprae. Chapter 3 describes the cloning, expression, purification and structural studies on SSBb from M. smegmatis (MsSSBb). MsSSBb has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. The protein crystallizes in hexagonal space group P6522 (a = b =73.61 Å, c = 216.21 Å), with half a tetrameric molecule in the asymmetric unit of the cell. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. A comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do. It was realized that many bacterial species have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation during sporulation. Chapter 4 describes the distinct properties and the role of SSBb in mycobacteria. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most bacterial SSB proteins, M. smegmatis SSBb (MsSSBb) forms a stable tetramer. However, solution studies indicate that MsSSBb is less stable towards thermal and chemical denaturation than MsSSBa. Also, in contrast to the 5-20 fold differences in DNA binding affinity between paralogous SSBs observed in other organisms, MsSSBb is only about twofold poorer in its DNA binding affinity than MsSSBa. The expression levels of ssbB gene increased during UV and hypoxic stresses, while the levels of ssbA expression declined. A direct physical interaction of MsSSBb and RecA, mediated by the C-terminal tail of MsSSBb was also established. The results obtained in this study indicate a role of MsSSBb in recombination repair during stress. Chapter 5 describes the characterization of the previously annotated hypothetical protein RecGwed and its probable role as a novel regulator in the resolution of branched DNA structures. The protein is composed of an unusually charged N-terminus and a C-terminal 'wedge' domain, similar to the wedge domain of RecG. A database search suggested that RecGwed is predominately present in the phylum Actinobacteria, along with some other known human pathogens. Purified M. smegmatis RecGwed (MsRecGwed) exists as a stable monomer in the solution. CD studies and homology modeling indicated an unusually low content of regular secondary structures. MsRecGwed was able to bind branched DNA structures such as Holliday junction, three-way junction, three-strand junction and replication fork in vitro, while it does not interact with ss- or dsDNA. The expression of recGwed in M. smegmatis was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2O2 treatment. These observations indicate the possibility of involvement of RecGwed in DNA transactions in post-replicative (stationary phase) recombination events, that proceed though branched DNA intermediates. The work described in this chapter is the first report of characterization of RecGwed-like proteins. Taken together, the work done in this thesis augments the existing repertoire of proteins known to be involved in DNA repair pathways in mycobacteria. As indicated in the concluding chapter, this study also creates a trail of future experiments that will improve our current understanding of HR in mycobacteria. As a part of ongoing efforts in the laboratory, on the characterization of enzymes which sanitize the nucleotide pool to prevent DNA damage, structural studies on M. smegmatis MutT2 have been carried out (Appendix). Structure of the native protein, and its complexes with substrates 5me-dCTP, dCTP and CTP and the respective products, has been determined. The work presented here is the first report of MutT2-type CTP pyrophosphorylase enzymes in complex with substrates. It provides insights into the mechanism of action and the molecular basis of the functioning of mycobacterial MutT2

Agrobacterium tumefaciens is a gram-negative soil bacterium that causes crown gall tumors on dicotyledenous plants. The transferred DNA (T-DNA) portion of the A. tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. The T-DNA is accompanied into plant cells by the VirD2 endonuclease covalently attached to its 5' end. VirE2, a cooperative, single-stranded DNA-binding protein is also transported into plant cells during infection by A. tumefaciens. VirD2 and VirE2 contain nuclear localization signals (NLSs) and are transported into the plant cell nucleus. The location of functional domains by the insertion of Xhol linker oligonucleotides throughout virE2 is reported. A ssDNA binding domain was located in the C-terminal half of VirE2, as well two domains involved in cooperative single-stranded DNA binding. Further, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity, but did not affect ssDNA binding.
Graduation date: 1997

碩士
國立清華大學
生物資訊與結構生物研究所
95
Abstract Single-stranded DNA binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair and recombination. The N-terminal domain of SSB forms an oligonucleotides/oligosaccharides binding (OB) fold, which function as single-stranded DNA (ssDNA) binding domain. The C-terminal conserved tail is supposed to participate in the protein-protein interaction. The crystal structure of C-terminal truncated SSB from Helicobacter pylori (residues 1-134, HpSSB134) bound to two dT(pT)34 [HpSSB134-dT(pT)34] was determined by molecular replacement method at 2.3 Å. The ssDNA wraps around four subunits of HpSSB134 by the electrostatic and hydrophobic stacking interactions. Four aromatic residues, Phe37, Phe50, Phe56 and Trp84, interact with the base of ssDNA by stacking arrangement. Meanwhile, four basic residues, Arg10, Arg36, Arg54 and Arg94, on the surface of HpSSB134 form a significant electrostatic path corresponding to the ssDNA binding.

碩士
國立清華大學
生物資訊與結構生物研究所
94
Single-stranded DNA binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination. SSB of Helicobacter pylori (HpSSB) is encoded by the ssb gene and contains 179 residues. The crystal structure of truncated HpSSB protein (residue 1-134) complexed with dT(pT)34 was determined at 3.1  resolution by X-ray crystallographic method. HpSSB exists as a tetramer in both crystal and solution states. The N-terminal domain (residue 1-115) contains an OB-fold (oligonucleotides binding fold), which is similar with other species like E. coli, to function as an ssDNA binding domain. However, the ssDNA binding mode of tmHpSSB134 exhibits a considerable variability with comparison to that of E. coli. In the structure of tmHpSSB134-dT(pT)34 complex, the ssDNA wraps on the OB-fold with mainly electrostatic and stacking interactions. Several basic residues, Arg10, Arg35, and Lsy108, on the surface of tmHpSSB134 form a significant patch to accommodate the ssDNA binding. Furthermore, two aromatic residues, Phe50 and Trp84, interact with thymidine by stacking interaction. The structure of residues 116-134 was unable to be determined because of its flexibility. Many evidences reveal that the acidic C-terminal tail of SSB might participate in the protein-protein interaction. The C-terminal interactions may trigger the activities of the associated proteins in DNA metabolism.

Single stranded DNA binding proteins (SSB) play a major role in cellular DNA processing events such as replication, recombination and repair and are central to maintaining the integrity of our genome. These processes require the DNA double helix to unwind, exposing less stable and highly vulnerable regions of single stranded DNA (ssDNA). SSB proteins bind ssDNA via a highly conserved oligonucleotide-binding (OB) domain and function to temporarily bind and protect exposed ssDNA generated during these events. The vital role of SSBs is evident from their ubiquitous presence in all forms of life. In the recent years, high resolution DNA-bound structures of bacterial SSBs and the eukaryotic Replication Protein A (RPA) were published, significantly enhancing our understanding of the molecular mechanism of DNA binding by SSBs. Although the structure of the archaeal SSB from Sulfolobus solfataricus (SsoSSB) has been solved, the DNA binding details of this protein have not been elucidated until now. This thesis reveals the structural basis of ssDNA recognition by SsoSSB and provides the first look into how archaeal SSBs bind ssDNA at the structural level. Two novel human SSBs, hSSB1 and hSSB2 were recently discovered. Prior to this, RPA was the only known SSB in humans, therefore this discovery has provided a new dimension to our understanding of DNA processing events in our cells and is now a prevailing topic of interest. The main function of the hSSBs appears to be central to a range of DNA repair pathways. However, irrespective of their precise function in DNA repair, both homologs are primarily involved in binding ssDNA, and act very early in the damage response. This has provided us with the opportunity to study hSSB1 and hSSB2 as suitable targets to shut down highly active DNA repair processes in tumour cells. In this thesis, I present the structural basis of DNA binding by hSSB1 and hSSB2 which will ultimately complement the development of hSSB inhibitors for the use in novel anti-cancer therapeutics.

Protozoan parasites of the genus Leishmania are the etiologic agents of a spectrum of important human diseases collectively referred to as leishmaniasis. The major surface protein on all species of Leishmania is a highly abundant 63 kDa glycoprotein referred to as GP63. GP63 has been characterized as a cell surface protease, however, its exact role in the Leishmania life cycle is not clear. The genes encoding GP63 are arranged in the Leishmania genome as a species-specific combination of direct head to tail tandem repeats and single dispersed gene copies. In the present study, a single repeat unit of the Leishmania donovani GP63 tandem array was cloned and sequenced. Alignment of the L. donovani GP63 gene sequence with the previously determined GP63 gene sequences from two related species, L. major and L. chagasi, revealed that GP63 is highly conserved across species. Consistent with the observed protease activity of GP63, the predicted amino acid sequence of GP63 from all three species contained a conserved motif shared by a number of zinc metalloproteases. In addition, alignment of the untranslated regions of the three GP63 genes revealed that the immediate 5' untranslated region is highly conserved within and across species. This region did not contain any sequences characteristic of higher eukaryotic promoter elements, however, it did contain an area of conserved hexanucleotide direct repeats. To determine whether these direct repeats (CTCGCC )represented a potential site of protein-DNA interaction, a A. gtl 1 expression library ofL. major was screened with a radio labelled oligonucleotide probe to detect clones expressing functional DNA-binding proteins. A gene was isolated which encoded a novel DNA-binding protein, referred to as HEXBP. The deduced amino acid sequence of HEXBP revealed that it is a 28 kDa protein containing nine 'CCHC-type' zinc finger motifs. The CCHC motif, Cys-X2_Cys-X4-His-X4-Cys, is invariant with regards to the number and spacing of cysteine and histidine residues and is shared by a number of nucleic acid-binding proteins. In accordance with the activity exhibited by other CCHC-containing proteins, HEXBP was characterized as a single-stranded nucleic acid-binding protein. Additional analyses indicated that HEXBP bound single-stranded DNA in a sequence specific manner and that the conserved 5' untranslated region of GP63 gene contained multiple HEXBP binding sites. To determine the cellular function of HEXBP, a HEXBP-deficient mutant of L.major was generated using the technique of double homologous gene replacement. Initial characterization of this mutant suggested that HEXBP was not essential for the expression of GP63 by in vitro cultivated promastigotes. Although the HEXBP-deficient mutant did not exhibit any gross phenotypic changes, further characterization will likely provide insight into the function of the HEXBP single-stranded DNA-binding protein. In addition, a plasmid construct was identified that lead to stable transformation of Leishmania when electroporated into promastigotes as an intact circular plasmid. This construct conferred selectable drug-resistance to transfectants and was found to replicate as an extrachromosomal circular concatamer. The construct was modified to produce a functional Leishmania expression vector called pLEX. Initial characterization of the transcriptional regulation of pLEX suggests that it also represents a potentially useful model system for studying the process of polycistronic gene expression in kinetoplastid protozoans.

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. The DNA replication process in RB69 (a T4-like bacteriophage) is similar to the processes in eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. In order to examine protein-protein interactions at the DNA replication fork, solution conditions have been established for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA and RB69 single-stranded DNA binding protein (gp32) using equilibrium fluorescence and light scattering. The interaction between DNA polymerase and single-stranded DNA binding protein has been characterized by fluorescence titrations and results in a 60-fold increase in the overall affinity of RB69 SSB for template-strand DNA in the presence of DNA polymerase. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site-of-action. A functional domain of RB69 single-stranded DNA-binding protein previously suggested to be the site of RB69 DNA polymerase:SSB interactions has been shown to be dispensable. The data from these studies have been used to model the RB69 DNA polymerase:SSB interaction at the primer-template junction. Fusion of RB69 SSB with its cognate DNA polymerase via a short six amino acid linker increases affinity for primer-template DNA by 6-fold and increases processivity by 7-fold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand entry channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

DNA is under constant attack from the external and internal environment. It is imperative to repair and maintain the vital genetic information of DNA. Otherwise it leads to an accumulation of mutations that alters the normal function of DNA and in turn causes a disorder in cellular metabolism. During repair, DNA unwinds into single-stranded DNA (ssDNA) and is even more vulnerable to damage. This is where human single-strand DNA binding protein 1 (hSSB1) binds and protects ssDNA. It is known that hSSB1 exists both as part of the MRN (MRE11, RAD50, NBS1) repair complex and the SOSS1 complex (made up of INTS3 and C9orf80). It is also known to initiate an appropriate repair mechanism by recruiting other proteins to the site of damage. There is an accumulating body of research on hSSB1 function in Double Strand Break Repair (DSBR). It promotes the ataxia telangiectasia mutated (ATM) kinase signalling cascade and also recruits the MRN complex to the site of double strand breaks (DSBs) in order to initiate DSBR via Homologous Recombination (HR). Recently, it was discovered that hSSB1 is capable of forming higher order oligomers and can function in the oxidative DNA damage response. The most common oxidative DNA damage is the 7,8-dihydro-8-oxoguanine (8-oxoG) adduct, which is the direct result of reactive oxygen species (ROS) produced during regular cellular respiration. If this damage goes unrepaired it may result in G:C to T:A transversions. These lesions are normally processed by the Base Excision Repair (BER) pathway, which involves human oxoguanine glycosylase (hOGG1) that cleaves the DNA backbone and removes the offending base. So far, it is understood that hSSB1 levels increase in response to oxidative damage; also, cells depleted of hSSB1 are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxoG adducts. The recruitment of hOGG1 to chromatin is dependent on hSSB1 and hSSB1 can promote hOGG1 cleavage of 8-oxoG. This thesis examines the mechanism of hSSB1 oligomerisation under oxidative conditions. hSSB1 forms dimers and tetramers and this oligomerisation is likely mediated by inter-domain disulfide bond formation. These oligomers can also be synthetically created through the use of a thiol reactive cross-linker. Oxidised hSSB1 binds to 8-oxoG damaged ssDNA with higher affinity than non-damaged ssDNA, likely indicating a direct role for oxidised hSSB1 in the recognition of 8-oxo-G lesions. Furthermore, hSSB1 and hOGG1 directly interact with a moderate binding affinity in the presence of 8-oxoG damaged ssDNA. Finally, a model of the tetramer is proposed using the recent crystal structure of monomeric hSSB1 as a template. The data presented here along with the proposed structural model allows hSSB1 to be placed in the oxidative DNA damage response pathway and gives crucial insight into the possible role of the oligomer in this process. As heightened levels of oxidative stress are associated with cancer (as well as aging and Alzheimer’s disease), understanding the molecular mechanisms of how cells repair oxidative DNA damage will be essential in the development of novel therapeutic treatments.

碩士
國立清華大學
生物科技研究所
97
Single stranded DNA (ssDNA) binding protein (SSB) plays essential roles in many processes related to DNA metabolism such as DNA replication, repair, and homologous genetic recombination. The HP1245 gene was annotated as SSB in Helicobacter pylori strain 26695. However, there are no functional or structured studies for this SSB up to now. The full length (179 residues) HP1245, a C-terminal truncated HP1245 containing 134 resides (tmHP1245 (1~134)), and four HP1245 mutants each containing 134 residues with a specific site mutant (F37A, F50A, F56A and W84A) were individually cloned into pQE30 vector and expressed in E. coli SG13009 previously in our lab. Applied basic gene cloning techniques and available plasmids, the gene containing full length HP1245 SSB with a point mutation on F37A, F50A, F56A or W84A was separately constructed and expressed in pQE30 containing E. coli SG13009 system. In addition, EcoSSB containing expression vector in E. coli SG13009 system was also prepared in this study. The binding activity between single stranded DNA and any one of the above mentioned fresh prepared HP1245 proteins (full length wild type, full length point mutants and C-terminal truncated mutant) were measured by means of (a) fluorescence titration (fixed amount of SSB plus fragmented calf thymus ss-DNA, or 600-1200 bp), (b) Electrophoresis mobility shift assay (EMSA) (SSB plus fixed amount of M13mp18 ssDNA) and (c) SPR (biotin-labeled d(T)35 ssDNA) in this study. The results of the fluorescence titration from the measurement of the tryptophan quenching due to the ssDNA binding to SSB provided one useful parameter, binding site sizes of nucleotides or Napp to wrap around (or cover) a SSB tetramer. Under high salt condition at 300 mM NaCl, the Napp for the full length HP1245 SSB was 35 + 2 nucleotides/tetramer, shorter than that for EcoSSB (61 + 4 NT/tetramer). The tmHP1245 owned the shortest Napp (30 + 1 NT/tetramer) among the various HP1245 SSB mutants used in this study. Similar results were also observed from SSB binding to ssDNA at low salt 10 mM NaCl condition, suggesting that the C-terminal of HP1245 SSB should play a role on ssDNA binding. On the other hand, EMSA results on retardation of the single stranded M13mp18 plasmid DNA migration on DNA agarose gel during SSB binding showed that about 330~439 molecules of full length HP1245 tetramer would saturate to bind one molecule of M13mp18 at high or low salt. Strong positive co-operativity showed in wild type HP1245 SSB-M13 complexes only at low salt condition. More tm-HP1245 tetramers were required to saturate the binding of single M13 molecule, indicating the C-terminal region of HP1245 affected the retardation of EMSA. The interaction of a series SSB with ssDNA has been further measured in real time by using a surface plasmon-resonance (SPR) and biosensor chip. Wild type HP1245 SSB was first applied onto the sensor chip surface that was pre-coated with Au, MUA, EDC/NHS, Streptoavidin and 5’-Biotinyl-poly(dT)35 in order. SPR measure at different conditions including: strpetavidin immobilization buffer pH value (Figure 11), 5’ biotinyl-poly(dT)35 capacity (Figure 12), flow rate of kinetic experiments (Figure13), regeneration buffer (Figure 14), HP1245 protein concentration and association time (Figure 16) were examined to obtain optimal conditions for further DNA binding experiments for each of above mentioned various HP1245 SSB. Response unit (RU) data from SPR after processing software program, BIAevaluation version 4.1 were transformed into useful parameters, such as ka, kd, KA, KD etc. to describe the SSB and ssDNA binding affinity. The KD of wild type HP1245 SSB binding poly dT 35mer was 0.16 nM in using BiacoreX (Figure 16A) and 0.1 nM in using Biacore3000 (Figure 16B), although different association time was used, 2 min for the former and 5 min for the latter. Higher KD (1.64 nM, about 9.9-fold, Figure 18) was obtained for C-terminal tm-HP1245 (1~134) in comparison with that from full-length HP1245 SSB to bind ssDNA. This result again emphasized that C-terminal of HP1245 was important for ssDNA binding. The importance of the C-terminal region on HP1245 was confirmed in results from fluorescence titration, EMSA and SPR measurement in this study. KD value for the binding of SSB to ssDNA from the lowest to highest is 0.16 nM for full length wild type HP1245 SSB, 1.6 nM (10-fold) for F37A mutant (Figure 19), 1.64 nM (10-fold) for tm-HP1245 (1~134) (Figure 18), 2.3 nM (14.1-fold) for W84A mutant (Figure 19), 3.06 nM (18.4-fold) for F50A mutant (Figure 19) and 3.12 nM (18.6-fold) for F56A mutant (Figure 20). These results suggested that the mutation of F37, F50, F56 or W84 in HP1245 SSB affected its binding to ssDNA, resulting in less KA (more binding affinity between SSB and ssDNA) or more KD (less dissociation for SSB-ssDNA complex) than that for wild type HP1245 SSB. Thus, SPR analysis was the most convenient method to examine the binding between ssDNA and different HP1245 SSB. It demonstrated that HP1245 SSB binds single stranded DNA with high affinity, by involving a tryptophan residue W84, and 3 phenylalanines F37, F50 and F56. Either SPR, fluorescence titration or EMSA could be used to distinguish different KD between tm-HP1245 SSB and wild type HP1245 SSB for ssDNA binding, higher KD (10-fold) in the former than that in the latter. This confirmed that the C-terminal region of HP1245 SSB was also important to bind ssDNA.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.
Dissertation

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus.
Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.