Gotowa bibliografia 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.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Physical Sciences<br>Science, Environment, Engineering and Technology<br>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.<br>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.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Environment and Sc<br>Science, Environment, Engineering and Technology<br>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.