Статті в журналах з теми "RAD51C/XRCC3"
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1
Somyajit, Kumar, Shivakumar Basavaraju, Ralph Scully, and Ganesh Nagaraju. "ATM- and ATR-Mediated Phosphorylation of XRCC3 Regulates DNA Double-Strand Break-Induced Checkpoint Activation and Repair." Molecular and Cellular Biology 33, no. 9 (February 25, 2013): 1830–44. http://dx.doi.org/10.1128/mcb.01521-12.
Повний текст джерелаАнотація:
The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G 2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G 2 /M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.
2
Yamada, Nazumi Alice, John M. Hinz, Vicki L. Kopf, Kathryn D. Segalle, and Larry H. Thompson. "XRCC3 ATPase Activity Is Required for Normal XRCC3-Rad51C Complex Dynamics and Homologous Recombination." Journal of Biological Chemistry 279, no. 22 (March 22, 2004): 23250–54. http://dx.doi.org/10.1074/jbc.m402247200.
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Hatanaka, Atsushi, Mitsuyoshi Yamazoe, Julian E. Sale, Minoru Takata, Kazuhiko Yamamoto, Hiroyuki Kitao, Eiichiro Sonoda, Koji Kikuchi, Yasukazu Yonetani, and Shunichi Takeda. "Similar Effects of Brca2 Truncation and Rad51 Paralog Deficiency on Immunoglobulin V Gene Diversification in DT40 Cells Support an Early Role for Rad51 Paralogs in Homologous Recombination." Molecular and Cellular Biology 25, no. 3 (February 1, 2005): 1124–34. http://dx.doi.org/10.1128/mcb.25.3.1124-1134.2005.
Повний текст джерелаАнотація:
ABSTRACT BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3′ end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.
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Nagaraju, Ganesh, Andrea Hartlerode, Amy Kwok, Gurushankar Chandramouly, and Ralph Scully. "XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids." Molecular and Cellular Biology 29, no. 15 (May 26, 2009): 4283–94. http://dx.doi.org/10.1128/mcb.01406-08.
Повний текст джерелаАнотація:
ABSTRACT Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.
5
Kurumizaka, H., S. Ikawa, M. Nakada, K. Eda, W. Kagawa, M. Takata, S. Takeda, S. Yokoyama, and T. Shibata. "Homologous-pairing activity of the human DNA-repair proteins Xrcc3*Rad51C." Proceedings of the National Academy of Sciences 98, no. 10 (May 1, 2001): 5538–43. http://dx.doi.org/10.1073/pnas.091603098.
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Wiese, C. "Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells." Nucleic Acids Research 30, no. 4 (February 15, 2002): 1001–8. http://dx.doi.org/10.1093/nar/30.4.1001.
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7
Liu, Yilun, Madalena Tarsounas, Paul O'Regan, and Stephen C. West. "Role of RAD51C and XRCC3 in Genetic Recombination and DNA Repair." Journal of Biological Chemistry 282, no. 3 (November 17, 2006): 1973–79. http://dx.doi.org/10.1074/jbc.m609066200.
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Masson, J. Y., A. Z. Stasiak, A. Stasiak, F. E. Benson, and S. C. West. "Complex formation by the human RAD51C and XRCC3 recombination repair proteins." Proceedings of the National Academy of Sciences 98, no. 15 (July 17, 2001): 8440–46. http://dx.doi.org/10.1073/pnas.111005698.
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Lio, Yi-Ching, David Schild, Mark A. Brenneman, J. Leslie Redpath, and David J. Chen. "Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells." Journal of Biological Chemistry 279, no. 40 (October 2004): 42313–20. http://dx.doi.org/10.1074/jbc.m405212200.
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10
Kurumizaka, H. "Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein." Nucleic Acids Research 31, no. 14 (July 15, 2003): 4041–50. http://dx.doi.org/10.1093/nar/gkg442.
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11
Abdu, Uri, Acaimo González-Reyes, Amin Ghabrial, and Trudi Schüpbach. "The Drosophila spn-D Gene Encodes a RAD51C-Like Protein That Is Required Exclusively During Meiosis." Genetics 165, no. 1 (September 1, 2003): 197–204. http://dx.doi.org/10.1093/genetics/165.1.197.
Повний текст джерелаАнотація:
Abstract In Drosophila, mutations in double-strand DNA break (DSB) repair enzymes, such as spn-B, activate a meiotic checkpoint leading to dorsal-ventral patterning defects in the egg and an abnormal appearance of the oocyte nucleus. Mutations in spn-D cause an array of ovarian phenotypes similar to spn-B. We have cloned the spn-D locus and found that it encodes a protein of 271 amino acids that shows significant homology to the human RAD51C protein. In mammals the spn-B and spn-D homologs, XRCC3 and RAD51C, play a role in genomic stability in somatic cells. To test for a similar role for spn-B and spn-D in double-strand DNA repair in mitotic cells, we analyzed the sensitivity of single and double mutants to DSBs induced by exposure to X rays and MMS. We found that neither singly mutant nor doubly mutant animals were significantly sensitized to MMS or X rays. These results suggest that spn-B and spn-D act in meiotic recombination but not in repair of DSBs in somatic cells. As there is no apparent ortholog of the meiosis-specific DMC1 gene in the Drosophila genome, and given their meiosis-specific requirement, we suggest that spn-B and spn-D may have a function comparable to DMC1.
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Su, Hang, Zhihao Cheng, Jiyue Huang, Juan Lin, Gregory P. Copenhaver, Hong Ma, and Yingxiang Wang. "Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination." PLOS Genetics 13, no. 5 (May 31, 2017): e1006827. http://dx.doi.org/10.1371/journal.pgen.1006827.
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13
Tarsounas, Madalena, Adelina A. Davies, and Stephen C. West. "RAD51 localization and activation following DNA damage." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1441 (January 29, 2004): 87–93. http://dx.doi.org/10.1098/rstb.2003.1368.
Повний текст джерелаАнотація:
The efficient repair of double–strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA–damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.
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Sullivan, Meghan R., and Kara A. Bernstein. "RAD-ical New Insights into RAD51 Regulation." Genes 9, no. 12 (December 13, 2018): 629. http://dx.doi.org/10.3390/genes9120629.
Повний текст джерелаАнотація:
The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.
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Simo Cheyou, Estelle, Jacopo Boni, Jonathan Boulais, Edgar Pinedo-Carpio, Abba Malina, Dana Sherill-Rofe, Vincent M. Luo, et al. "Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability." PLOS Genetics 18, no. 11 (November 14, 2022): e1010495. http://dx.doi.org/10.1371/journal.pgen.1010495.
Повний текст джерелаАнотація:
Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.
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Castro, Michael, Ansu Kumar, Himanshu Grover, Vivek Patil, Ashish Agrawal, Anuj Tyagi, Humera Azam, et al. "Cellworks Omics Biology Model (CBM) to predict therapy response and identify novel biomarkers for 5FU-based combination therapy in gastric cancer patients." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e16091-e16091. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e16091.
Повний текст джерелаАнотація:
e16091 Background: Using a 5FU backbone, a variety of combination chemotherapy regimens have been adopted for gastric cancer. However, the genetic heterogeneity of the disease suggests certain drugs would lead to better outcomes for specific patients. In principle, the incorporation of molecular profiling information into treatment selection can generate superior survival for patients. Therefore, we conducted a pilot study using the CBM to identify novel genomic biomarkers of response and resistance to several 5FU-based regimens. Methods: 12 gastric cancer patients treated with 5FU-cisplatin (N = 4), 5FU-VP16 (N = 5) and 5FU-cisplatin-docetaxel (N = 3) were selected from the TCGA database. Mutation and copy number aberrations from each case served as input for CBM to generate patient-specific protein network maps generated from PubMed and other resources. Disease-biomarkers unique to each patient were identified within protein network maps. Drug impact on the disease network was digitally simulated to determine treatment efficacy by measuring effect of chemotherapy on the cell growth score, i.e., a composite of cell proliferation, viability, apoptosis, metastasis, DNA damage and other cancer hallmarks. Effectively, the mechanism of action of each drug was mapped to each patient’s genome and the biological consequences of genomic abnormalities were correlated with response. Results: Of the 12 patients in the study, 10 patients responded to 5FU-based regimens and 2 were non-responders. CBM correctly predicted the therapy response in each therapy segment. Key response criteria were: 5FU-cisplatin - ARID1A (LOF), RAD51 (LOF), MBD1 (LOF), DUT (LOF). 5FU-VP16 - FBXW7 (DEL), RAD51D (DEL), RAD51C (DEL), XRCC2 (DEL), XRCC3 (DEL). 5FU-cisplatin-docetaxel - ARID1A (DEL), CHEK1 (DEL), REV3L (DEL), XRCC3(DEL) and TUBB (AMP). Resistance biomarkers were also identified for these drugs. FBXW7 loss caused cisplatin resistance, but also predicted sensitivity to VP16. Interestingly, all five 5FU-VP16 responders harbored FBXW7 loss. Similarly, in two 5FU-cisplatin-docetaxel non-responders, KIFC1 (GOF), YBX1(AMP), CDC20 (AMP) were key biomarkers for docetaxel resistance. In one of these patients, ATM (DEL), RBBP8 (DEL) and GSTP1 (DEL) predicted response to VP16. Conclusions: The efficacy of individual cytotoxic agents varies in the population. Mutations and copy number changes in DNA checkpoint, DNA repair, and tumor suppressor genes impact responsive to chemotherapy and should be routinely assessed. Notably, 5FU-VP16 combination could have delivered better antitumor efficacy for several non-responders or 25% of the patients in this small cohort. CBM is able to identify both the most active as well inefficacious components of the treatment upfront. We suggest that this approach should be prospectively evaluated in a larger cohort.
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Alagpulinsa, David, Srinivas Ayyadevara, Shmuel Yaccoby, and Robert shmookler Reis. "A Peptide Nucleic Acid Targeting Nuclear Rad51 Sensitizes Myeloma Cells to Melphalan Chemotoxicity Both in Vitro and in Vivo." Blood 124, no. 21 (December 6, 2014): 3529. http://dx.doi.org/10.1182/blood.v124.21.3529.3529.
Повний текст джерелаАнотація:
Abstract Multiple myeloma (MM) cells are characterized by extensive genomic heterogeneity, which contributes to patient differences in prognosis and response to treatment. We previously reported that MM cells have elevated homologous recombination (HR) rates and expression of RAD51 and its paralogs, promoting genomic instability and disease progression that are reversed by RAD51 siRNA. We now examine the roles of HR and RAD51 in resistance to melphalan, one of the most widely used drugs for MM chemotherapy. The drug induces a variety of DNA lesions, with DNA interstrand crosslinks (ICL) accounting for most of the drug’s cytotoxicity. RAD51 is a central protein in the HR pathway and its overexpression may contribute to chemoresistance by enabling repair of DNA lesions induced by DNA damaging agents such as melphalan. MM cell sensitivity to melphalan correlates directly with melphalan-induced RAD51 foci, and high RAD51 expression predicts poor event-free and overall survival of MM patients. Activity of the Rad51 promoter increases >850-fold in cancer cells compared to normal cells, and tumor cells are selectively killed by a construct in which PRad51 drives expression of diphtheria toxin. In this study, we tested whether inhibiting RAD51 expression with a peptide nucleic acid (PNA) would inhibit MM cell growth and/or sensitize MM cells to melphalan. PNAs are DNA or RNA mimics in which a polymer of (2-aminoethyl) glycine replaces the nucleic acid’s sugar-phosphate backbone. PNAs are highly specific, binding DNA with higher affinity than RNA or DNA, and they are quite stable to degradation both in vitro and in vivo. We designed a PNA to target the promoter region of the RAD51 gene (PNArad51), encompassing the transcription start site. To enhance cellular uptake and nuclear delivery without transfection, we conjugated the PNA to a nuclear localization signal rich in basic residues (PKKKRKVR). As a control we employed a scrambled PNA (PNAmt) with the same nucleotide composition but not targeting any genomic sequences. We used qRT-PCR to assess the effect of PNA on RAD51 mRNA expression and that of melphalan on mRNA levels of RAD51 and its paralogs (RAD51B, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and BRCA1. Propidium iodide staining and flow cytometry were used to examine the cell-cycle effects of melphalan. γH2AX and RAD51 foci were quantitated using confocal immunofluorescence microscopy, and MM cell viability was assessed with the WST-1 assay. To examine the in vivo consequences of PNA ± melphalan for tumor growth, we injected H929 MM cells expressing luciferase into rabbit bone fragments implanted in SCID-rab mice, as previously described by us,. Total RNA extracted from cells recovered from the rabbit bones was analyzed by qRT-PCR to determine the in vivo effect of PNA on expression of RAD51. Melphalan treatment (10 µM) significantly induced expression of RAD51 and its paralogs, particularly RAD51 and XRCC3 (p≤0.01). Melphalan caused cell-cycle arrest, predominantly in the S-phase (55%, significantly elevated over vehicle alone, 17%; p<0.0001), the period in which HR is most active, and during which ICLs are converted into double strand breaks (DSBs) on encountering DNA replication forks. PNArad51 (10 µM) significantly reduced expression of RAD51 (~60%, p<0.001) relative to PNAmt. Pretreatment with PNArad51 inhibited melphalan-induced RAD51 focus formation, far more than PNAmt pretreatment (21% compared to 66%, p<0.0001) whereas the number of γH2AX foci increased (66%) relative to PNAmt (39%; p<0.0001). Consequently, pretreatment with PNArad51 produced synergistic synthetic lethality with melphalan, reducing the IC50 of melphalan by 4.5-fold. PNArad51 alone caused significant cytotoxicity compared to PNAmt (p<0.05). In the SCID-rab mouse model, a two-week treatment with PNArad51 alone or in combination with melphalan resulted in significant inhibition of tumor volume (p≈0.01 and p<0.05, respectively) compared to PNAmt, although the combination of PNAmt plus melphalan was ineffectual. Prolonged treatment (4 weeks) with PNArad51 ± melphalan (but not PNAmt + melphalan) reduced tumor growth compared to PNAmt treatment, although this was not statistically significant (p>0.05). These results highlight the importance of RAD51 in the response of MM cells to melphalan, and indicate for the first time the potential for RAD51-targeted PNA in tumor chemosensitization. Disclosures No relevant conflicts of interest to declare.
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Kim, So Hyeon, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, et al. "Abstract LB228: Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells." Cancer Research 83, no. 8_Supplement (April 14, 2023): LB228. http://dx.doi.org/10.1158/1538-7445.am2023-lb228.
Повний текст джерелаАнотація:
Abstract Background: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinases, which is approved for HER2-positive metastatic breast cancer as second line treatment. Significant proportion of patients experience disease progression due to acquired resistance. Activation of DNA damage repair (DDR) is one of the drug-resistance mechanism, however, the impact of DDR on sensitivity to lapatinib is unclear. Thus, lapatinib resistance mechanism was explored from the standpoint of DDR activation. Methods: Acquired lapatinib-resistant (LR) SK-BR-3 cell lines were generated by continuously exposing to lapatinib, starting with 30 nmol/L and incrementally increasing to 10 μmol/L over 7 months. MTT assay was used to confirm lapatinib sensitivity. Cell cycle progression was analyzed using BrdU assay. Replication fork speed and stalled fork were analyzed by DNA fiber assay. Expression of the molecules was examined using Western blotting, immunofluorescence assay and transcriptome data analysis. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. Results: G1/S phase transition was increased and early/late S phase population was increased in SK-BR-3 LR cells. Replication fork speed was accelerated and replication stress are elevated in SK-BR-3 LR cells. p-Chk1, Rad51, Rad51B, Rad51C and XRCC3 were upregulated in SK-BR-3 LR cells. After irradiation or treatment with ATR inhibitor, γ-H2AX was continuously increased in parental cells. In contrast, γ-H2AX did not change significantly in SK-BR-3 LR cells, and Chk1 was activated after irradiation or treatment with ATR inhibitor. Tail of comet disappeared at early time point in in SK-BR-3 LR cells after induction of DNA damage compared with parental cells. These results demonstrated that DDR was activated and DNA damage repair capacity was enhanced in LR cells. Conclusion: Replication stress is elevated in SK-BR-3 LR cells. Up-regulation of molecules involved in the homologous recombination repair pathway was observed in SK-BR-3 LR cells. Moreover, DNA repair capacity was increased in SK-BR-3 LR cells. These data suggested that activation of DNA damage repair pathway caused by replication stress contributes to lapatinib resistance. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, Changyun Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB228.
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Sinha, Asha, Ali Saleh, Raelene Endersby, Shek H. Yuan, Chirayu R. Chokshi, Kevin R. Brown, Bozena Kuzio, et al. "RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status." Cancers 12, no. 11 (October 29, 2020): 3178. http://dx.doi.org/10.3390/cancers12113178.
Повний текст джерелаАнотація:
PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.
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Ding, Yan, Can-Lan Sun, Liton Francisco, Melanie Sabado, Liang Li, Brian Hahn, Garrett Larson, Stephen J. Forman, Ravi Bhatia, and Smita Bhatia. "Genetic Susceptibility to Therapy-Related Leukemia (t-MDS/AML) After Hodgkin Lymphoma (HL) or Non-Hodgkin Lymphoma (NHL)." Blood 114, no. 22 (November 20, 2009): 199. http://dx.doi.org/10.1182/blood.v114.22.199.199.
Повний текст джерелаАнотація:
Abstract Abstract 199 Therapy-induced t-MDS/AML is the leading cause of non-relapse mortality after HL or NHL. However, there exists a wide variation in t-MDS/AML susceptibility – potentially explained by individual variability in drug metabolism, DNA repair and apoptosis, or in genetic profiles shared with de novo AML. Using a matched case-control study design, we examined the association between t-MDS/AML and candidate genes (n=29) in relevant biological pathways, including hematopoietic regulation (RUNX1, HLX1); apoptosis (TP53, MDM2); drug metabolism (CYP3A4, CYP1A1, GSTM1, GSTP1, GSTT1, NQO1); DNA repair (MGMT, MUTYH, MLH1, MSH2, MTHFR, RAD51, RAD51C, RAD52, XRCC1, XRCC2, XRCC3, XRCC4, XPD); and genes associated withde novoAML (LAMC2/NMNAT2, SGCE/PEG10, FRAP1, and PTPRT). The study cohort consisted of 46 cases with t-MDS/AML after HL/NHL and 46 controls with HL/NHL, but without t-MDS/AML (matching criteria: primary diagnosis, age and year of primary diagnosis, length of follow-up and genetic ancestry [see Table]). Sequenom MassArray and PCR were used to analyze 55 SNPs and 2 deletions in these genes. After correction for multiple testing, we could not identify significant association between any single SNP and t-MDS/AML. However, we did confirm enrichment of risk genotypes in t-MDS/AML for 2 of 4 loci previously implicated in de novo AML by a genome wide association study in Caucasians: LAMC2/NMNAT2 (minor allele carrier [MAC] odds ratio [OR]=4.0, p=0.1) and PTPRT (MAC OR=4.5, p=0.06). Next, we tested the hypothesis that TP53 plays a role in mediating apoptotic response to DNA damage following genotoxic exposures. We modeled interactions between a common coding SNP of TP53 causing a Pro 72 to Arg variant (P72R: Pro allele is associated with ∼15-fold decreased apoptotic capacity compared to Arg) and other candidate SNPs. We identified significant interaction between TP53 and several SNPs in MTHFR (Min. likelihood ratio pinteraction=0.0003 and 0.04 adjusted after 10000 permutations). Although the homozygous T allele of rs7538516 (associated with lower MTHFR expression) was not associated with t-MDS/AML by itself, it increased the risk 11-fold (OR=11.4, p=0.005) when combined with a Pro/Arg or Pro/Pro genotype of P72R compared to its combination with Arg/Arg. This observation suggests that reduced MTHFR activity (associated with increased risk of chromosomal aberrations during DNA repair), in combination with reduced apoptotic capacity (Pro/Arg or Pro/Pro variant of TP53), increases the risk of t-MDS/AML. Next, we examined expression levels of these 29 genes (79 probe sets in Affymetrix U133 Plus 2.0 microarrays) in a subset of 13 cases matched with 28 controls. Using a general linear model adjusted for age, gender, race, and exposure to alkylating agents, topoisomerase inhibitors and radiation, we detected 11 of the 29 (38%) genes to be differentially expressed between cases and controls (p<0.05). Strong signals (p<0.05 after Holm-Bonferroni adjustment) were observed for TP53 (apoptosis), genes involved in drug metabolism (CYP3A4 [activation], GSTM1, GSTP1, and GSTT1 [detoxification]), in DNA repair (MSH2), and genes associated with de novo MDS/AML (NMNAT2, PEG10). Taken together, these observations provide evidence supporting the following: i) a significant association between individual capacity of apoptosis, drug metabolism, DNA repair, and t-MDS/AML; and ii) shared genetic susceptibility between t-MDS/AML and de novo AML. These observations not only further our understanding of the pathogenesis of t-MDS/AML, but also help identify those at the highest risk, setting the stage for targeted surveillance or pharmacological interventions. Disclosures: No relevant conflicts of interest to declare.
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Alolayan, Ashwaq, Fouad Sabatin, Mohammed algarni, Nadine Mabsout, Horya Zaher, Hussam Shehata, Saeed Alturki, et al. "Abstract P6-02-01: Frequency of pathogenic germline mutations beyond Germline BRCA gene mutations among Saudi patients with breast cancer." Cancer Research 83, no. 5_Supplement (March 1, 2023): P6–02–01—P6–02–01. http://dx.doi.org/10.1158/1538-7445.sabcs22-p6-02-01.
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Abstract Frequency of pathogenic germline mutations beyond Germline BRCA gene mutations among Saudi patients with breast cancer Mohammed Algarni*1,2,3, Ashwaq Alolayan1,2,3, Fouad Sabatin1,2,3, Nadine Mabsout1, Horya Zaher1, Hussam Shehata1, Saeed Alturki4, Abdulaziz alshalhoub1, Fatimah Alturki1, Sadal Refaea1,2,3, Nafisah Abdelhafiz1,2,3, Turki Alfayea 1,2,3, Mohammed Al Balwi1,2,3 Kanan Alshammari1,2,3 1King Abdulaziz Medical City, Ministry of National Guard – Health Affairs, Riyadh, Saudi Arabia, 2King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia, 3King Abdullah International Medical Research Center, Riyadh, Saudi Arabia 4Anwa lab Riyadh, Saudi Arabia Background : Breast cancer is the commonest cancer diagnosed in the kingdom of Saudi Arabia. Although the majority of breast cancer cases are sporadic, around 25-30% are related to hereditary and familial components. Germline BRCA gene mutations are most common mutations associated with hereditary breast cancer predisposition syndromes. In Saudi Arabia, the reported frequency of germline BRCA mutations is 11%1,2. There is no data about the prevalence non BRCA pathogenic germline mutations in Saudi population. We aimed to study the prevalence of these mutations in Saudi patients with breast cancer. Methods : We analyzed all the confirmed breast cancer cases who were referred to the cancer genetic clinic at King Abdulaziz medical city in Riyadh, Kingdom of Saudi Arabia by using our cancer genetics database. Since November 2018, a comprehensive hereditary cancer gene panel is offered to all referred breast cancer cases who meet the NCCN testing guidelines after obtaining a genetic counselling assessment and an informed consent. All testing was internally funded by the institution.The comprehensive panel tested genes are; ABRAXAS1, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, DICER1, DIS3L2, EPCAM, FANCC, FH, FLCN, GALNT12, HNF1B, HOXB13, KIT, MC1R, MEN1, MET, MITF, MLH1, MLH3, MRE11, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NTHL1, PALB2, PMS1, PMS2, POLD1, POLE, POT1, PRSS1, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RECQL, RET, RNF43, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, STK11, TGFBR2, TP53, TSC1, TSC2, VHL, WT1, XRCC2, XRCC3. Result : Between November 2018 and May 2022, a total of 332 patients with breast cancer have been tested. The median age was 45 and 54 years for females and males, respectively. The majority of patients were females (n=322, 97%). Most of the patients had stage III disease (n=183, 55%) followed by stage II(n=91, 27%). Pathogenic variant(PVs) was reported in 16% (n= 52), variant of uncertain significance (VUSs) was reported in 10% (n=32) while no mutation reported in the rest of the patients. TNBC was the most common phenotype among carriers of pathogenic mutation (50%). The PVs reported were BRCA1 (n= 19),BRCA2 (n= 21), PALB2(n= 2), PTEN (n= 2), ATM(n= 1), BARD1(n= 1), BLM(n= 1), BRIP1(n= 1), CDKN2A(n= 1), CHECK2(n= 1), MSH2(n= 1) and RECQL(n= 1). Conclusion : This study shows that extended panel testing beyond BRCA gene increases the rate of detection of pathogenic germline mutations that has preventative and possibly therapeutic implications. In addition, to the best of our knowledge this is the first study that gives insight about the frequency of non germline BRCA mutations which represent unmet needs for breast cancer patients in Saudi Arabia. 1. J Glob Oncol. 2018 Aug;4:1-9. 2. Breast Cancer Res Treat. 2018 Apr;168(3):695-702 Citation Format: Ashwaq Alolayan, Fouad Sabatin, Mohammed algarni, Nadine Mabsout, Horya Zaher, Hussam Shehata, Saeed Alturki, Abdulaziz Alshalhoub, Fatimah Alturki, Sadal Refaea, Nafisah Abdelhafiz, Turki Alfayea, Kanan Alshammari, Mohammed Albalwi. Frequency of pathogenic germline mutations beyond Germline BRCA gene mutations among Saudi patients with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-01.
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Slupianek, Artur, Shuyue Ren, and Tomasz Skorski. "Selective Anti-Leukemia Targeting of the Interaction Between BCR/ABL and Mammalian RecA Homologs." Blood 112, no. 11 (November 16, 2008): 195. http://dx.doi.org/10.1182/blood.v112.11.195.195.
Повний текст джерелаАнотація:
Abstract We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.
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Mpakou, Vassiliki, Evangelia Papadavid, Evi Konsta, Vikentiou Murofora, Frieda Kontsioti, Sotiris Papageorgiou, Dikea-Eleni Ioannidou, et al. "Bortezomib and Methotrexate Interfere with the DNA Repair Signaling Transduction Pathways and Induce Apoptosis in Cutaneous T-Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 5232. http://dx.doi.org/10.1182/blood.v124.21.5232.5232.
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Abstract Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the commonest types and together comprise 54% of all CTCL. MF evolves from patches to infiltrated plaques and eventually tumors. SS is a lymphoma-leukemia syndrome characterized by erythroderma and the presence of a malignant T-cell clone in the peripheral blood and the skin. At present, no curative treatment for CTCL is available. Therefore current CTCL research efforts are focused on elucidating the molecular mechanisms of the disease’s pathogenesis and on identifying new pharmacological targets. Several drugs have shown potentially significant activity either alone or in combination with conventional agents. Their effectiveness and their mechanisms of action comprise a current research challenge for the improvement of CTCL therapy. The aim of this study was to investigate the possible alterations in the gene expression profile (focusing on DNA Damage Signaling and DNA Repair pathways) and cell death in CTCL cell lines after treatment with two chemotherapeutic agents, Bortezomib and Methotrexate. Methods: Three CTCL cell lines were used. MyLa, (MF), SeAx and Hut-78 (both SS). Cells were cultured in RPMI 1640 and were treated with either Bortezomib (10nmol/L) or Methotrexate (10μM) for 24h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. Gene expression profiling following PCR arrays analysis was performed after total RNA extraction and purification from untreated and drug-treated cells. All RNA samples’ amplification, labeling and hybridization to RT2 Profiler PCR Arrays (DNA Damage Signaling and DNA Repair PCR array) (QIAGEN) were performed according to the manufacturer’s instructions. All data were analyzed using the appropriate RT2 Profiler PCR Array data analysis tool. Results: Hut-78, Seax and Myla cells responded with statistically significant enhanced apoptosis when treated for 24h with bortezomib, compared to untreated cells, while Methotrexate led to a rather moderate increase of apoptosis in Hut-78 and Seax cells and did not affect the apoptosis of Myla cells. Microarrays analysis after bortezomib treatment revealed a great effect in the expression profile of genes involved in almost all DNA repair pathways tested, in all three cell lines, with Hut-78 being affected the most. Specifically, in all cell lines, there was a significant down-regulation of a large number of genes involved in the Double Strand Breaks DNA Repair mechanism, (i.e. BRCA1, BRCA2, RAD50, RAD51, RAD51C, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6) as well as of genes involved in the Mismatch Repair pathway (i.e. MLH1, MLH3, MSH2, MSH5, MSH6) and the Nucleotide Excision Repair mechanism (i.e. DDB2, LIG1 and RAD23A), compared to untreated cells. On the contrary, bortezomib had a small effect on Base Excision repair mechanism, mostly downregulating the expression of XRCC1 gene in Hut-78 and Myla cells. Methotrexate treatment also led to a significant down-regulation of genes involved in the DSB (RAD50, XRCC4, XRCC6), MMR (MSH4) and NER (CDK7, RAD23A) repair mechanisms in Hut-78 cells but had a rather much more moderate effect on the expression profile of Seax and Myla cells, where fewer genes were affected. Conclusions: Our data clearly demonstrate a differential effect of bortezomib and methotrexate in terms of apoptosis induction on CTCL cells with bortezomib inducing apoptosis of both MF and SS derived cell lines and methotrexate being rather inactive on SS derived cells. We showed that both drugs, but mostly bortezomib significantly down-regulate a large number of genes involved in the DSB, MMR and NER mechanisms, suggesting a possible mechanism, among probably others, for the enhanced sensitivity to apoptosis of SS and MF cell lines after treatment. Bortezomib’s significant effect could be easily understood, since it is a well known proteasome inhibitor and has been previously related to inhibition of NF-kB and accumulation of pro-apoptotic proteins, while it has also been reported that cancer cells are more sensitive to proteasome inhibition than normal cells. Although these results need to be further confirmed, they appear very encouraging for understanding the mechanisms of action of these drugs in CTCL with the view to ameliorate their use in clinical practice. Disclosures No relevant conflicts of interest to declare.
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Mishra, Anup, Sneha Saxena, Anjali Kaushal, and Ganesh Nagaraju. "RAD51C/XRCC3 Facilitates Mitochondrial DNA Replication and Maintains Integrity of the Mitochondrial Genome." Molecular and Cellular Biology 38, no. 3 (November 20, 2017). http://dx.doi.org/10.1128/mcb.00489-17.
Повний текст джерелаАнотація:
ABSTRACT Mechanisms underlying mitochondrial genome maintenance have recently gained wide attention, as mutations in mitochondrial DNA (mtDNA) lead to inherited muscular and neurological diseases, which are linked to aging and cancer. It was previously reported that human RAD51, RAD51C, and XRCC3 localize to mitochondria upon oxidative stress and are required for the maintenance of mtDNA stability. Since RAD51 and RAD51 paralogs are spontaneously imported into mitochondria, their precise role in mtDNA maintenance under unperturbed conditions remains elusive. Here, we show that RAD51C/XRCC3 is an additional component of the mitochondrial nucleoid having nucleus-independent roles in mtDNA maintenance. RAD51C/XRCC3 localizes to the mtDNA regulatory regions in the D-loop along with the mitochondrial polymerase POLG, and this recruitment is dependent upon Twinkle helicase. Moreover, upon replication stress, RAD51C and XRCC3 are further enriched at the mtDNA mutation hot spot region D310. Notably, the absence of RAD51C/XRCC3 affects the stability of POLG on mtDNA. As a consequence, RAD51C/XRCC3-deficient cells exhibit reduced mtDNA synthesis and increased lesions in the mitochondrial genome, leading to overall unhealthy mitochondria. Together, these findings lead to the proposal of a mechanism for a direct role of RAD51C/XRCC3 in maintaining mtDNA integrity under replication stress conditions.
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Longo, Michael A., Sunetra Roy, Yue Chen, Karl-Heinz Tomaszowski, Andrew S. Arvai, Jordan T. Pepper, Rebecca A. Boisvert, et al. "RAD51C-XRCC3 structure and cancer patient mutations define DNA replication roles." Nature Communications 14, no. 1 (July 24, 2023). http://dx.doi.org/10.1038/s41467-023-40096-1.
Повний текст джерелаАнотація:
AbstractRAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5’ RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.
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Prakash, Rohit, Yashpal Rawal, Meghan R. Sullivan, McKenzie K. Grundy, Hélène Bret, Michael J. Mihalevic, Hayley L. Rein, et al. "Homologous recombination–deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants." Proceedings of the National Academy of Sciences 119, no. 38 (September 13, 2022). http://dx.doi.org/10.1073/pnas.2202727119.
Повний текст джерелаАнотація:
Mutations in homologous recombination (HR) genes, including BRCA1 , BRCA2 , and the RAD51 paralog RAD51C , predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.
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AHLAWAT, SONIKA, REKHA SHARMA, REENA ARORA, LATIKA JAISWAL, MEENU CHOPRA, PRIYANKA SHARMA, and SACHINANDAN DE. "Conserved architecture of RAD51 recombinase in ruminants revealed through molecular cloning and characterization." Indian Journal of Animal Sciences 86, no. 12 (December 20, 2016). http://dx.doi.org/10.56093/ijans.v86i12.65979.
Повний текст джерелаАнотація:
Meiotic recombination is a highly conserved process that ensures accurate segregation of homologous chromosomes and contributes to genetic variability of a species to with stand the pressure of natural selection. RAD51 recombinase plays a pivotal role in double strand break repair during homologous recombination and also ensures that chromosomal integrity is maintained during meiotic cell cycle. The present study was aimed to clone and sequence characterize RAD51 gene from cDNA isolated from testicular tissue of four ruminant species (buffalo, cattle, sheep and goat). The complete open reading frame of RAD51 gene was observed to be 1020 nucleotides in length encoding a putative protein of 339 amino acids. Functional feature prediction by SMART and Pfam revealed highly conserved DNA binding h4elix-hairpin-helix motif in the N terminal domain and Walker A and Walker B motifs in the catalytic domain which confer ATP binding and hydrolysis activity, respectively. All RAD51 orthologs and paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) were subjected to phylogenetic analysis. Evolutionary analysis between the different orthologs and paralogs suggested that these proteins are conserved from yeast to humans since all the orthologs from different species clustered in their respective clade, clearly demarcating the paralogs. Gene duplication events have expanded the RAD51 family in eukaryotes offering them increased capacity to repair DNA and promote homologous recombination.