Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

Nature Communications ◽

10.1038/s41467-021-21354-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

David D. Shock ◽

William A. Beard ◽

Samuel H. Wilson

Keyword(s):

Active Site ◽

Dna Polymerases ◽

Time Lapse ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

X Ray Crystallography ◽

Polymerase Fidelity ◽

Break Repair

AbstractOxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

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A unique pathway of double-strand break repair operates in tandemly repeated genes.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1222 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1222-1231 ◽

Cited By ~ 85

Author(s):

B A Ozenberger ◽

G S Roeder

Keyword(s):

Gene Product ◽

Gene Array ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Yeast Saccharomyces Cerevisiae ◽

Spontaneous Recombination ◽

Genome Recombination ◽

Repeat Units ◽

Almost All

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

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PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713912115 ◽

2018 ◽

Vol 115 (8) ◽

pp. E1759-E1768 ◽

Cited By ~ 14

Author(s):

Jung-Kuei Chen ◽

Wen-Ling Lin ◽

Zhang Chen ◽

Hung-wen Liu

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Active Involvement ◽

Parp 1

Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events. Here we report cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous end-joining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. Taken together, these results reveal a previously unrecognized role for CIRBP in DSB repair.

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Double strand break (DSB) repair pathways in plants and their application in genome engineering

Genome editing for precision crop breeding ◽

10.1201/9781003048237-3 ◽

2021 ◽

pp. 27-61

Author(s):

Valerie Miller ◽

Natalja Beying ◽

Carla Schmidt ◽

Holger Puchta

Keyword(s):

Genome Engineering ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Repair Pathways

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Second-Generation Antiandrogen Therapy Radiosensitizes Prostate Cancer Regardless of Castration State through Inhibition of DNA Double Strand Break Repair

Cancers ◽

10.3390/cancers12092467 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2467 ◽

Cited By ~ 1

Author(s):

Mohamed E. Elsesy ◽

Su Jung Oh-Hohenhorst ◽

Anastassia Löser ◽

Christoph Oing ◽

Sally Mutiara ◽

...

Keyword(s):

Prostate Cancer ◽

First Generation ◽

Second Generation ◽

Strand Break ◽

Double Strand Break ◽

Abiraterone Acetate ◽

Double Strand Break Repair ◽

Dsb Repair ◽

Castration Resistant ◽

Break Repair

(1) Background: The combination of the first-generation antiandrogens and radiotherapy (RT) has been studied extensively in the clinical setting of prostate cancer (PCa). Here, we evaluated the potential radiosensitizing effect of the second-generation antiandrogens abiraterone acetate, apalutamide and enzalutamide. (2) Methods: Cell proliferation and agarose-colony forming assay were used to measure the effect on survival. Double strand break repair efficiency was monitored using immunofluorescence staining of γH2AX/53BP1. (3) Results: We report retrospectively a minor benefit for PCa patients received first-generation androgen blockers and RT compared to patients treated with RT alone. Combining either of the second-generation antiandrogens and 2Gy suppressed cell growth and increased doubling time significantly more than 2Gy alone, in both hormone-responsive LNCaP and castration-resistant C4-2B cells. These findings were recapitulated in resistant sub-clones to (i) hormone ablation (LNCaP-abl), (ii) abiraterone acetate (LNCaP-abi), (iii) apalutamide (LNCaP-ARN509), (iv) enzalutamide (C4-2B-ENZA), and in castration-resistant 22-RV1 cells. This radiosensitization effect was not observable using the first-generation antiandrogen bicalutamide. Inhibition of DNA DSB repair was found to contribute to the radiosensitization effect of second-generation antiandrogens, as demonstrated by a significant increase in residual γH2AX and 53BP1 foci numbers at 24h post-IR. DSB repair inhibition was further demonstrated in 22 patient-derived tumor slice cultures treated with abiraterone acetate before ex-vivo irradiation with 2Gy. (4) Conclusion: Together, these data show that second-generation antiandrogens can enhance radiosensitivity in PCa through DSB repair inhibition, regardless of their hormonal status. Translated into clinical practice, our results may help to find additional strategies to improve the effectiveness of RT in localized PCa, paving the way for a clinical trial.

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Involvement of POLA2 in Double Strand Break Repair and Genotoxic Stress

International Journal of Molecular Sciences ◽

10.3390/ijms21124245 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4245

Author(s):

Tuyen T. Dang ◽

Julio C. Morales

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Dna Polymerase ◽

Genotoxic Stress ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

Dna Polymerase Alpha ◽

Break Repair

Cellular survival is dependent on the efficient replication and transmission of genomic information. DNA damage can be introduced into the genome by several different methods, one being the act of DNA replication. Replication is a potent source of DNA damage and genomic instability, especially through the formation of DNA double strand breaks (DSBs). DNA polymerase alpha is responsible for replication initiation. One subunit of the DNA polymerase alpha replication machinery is POLA2. Given the connection between replication and genomic instability, we decided to examine the role of POLA2 in DSB repair, as little is known about this topic. We found that loss of POLA2 leads to an increase in spontaneous DSB formation. Loss of POLA2 also slows DSB repair kinetics after treatment with etoposide and inhibits both of the major double strand break repair pathways: non-homologous end-joining and homologous recombination. In addition, loss of POLA2 leads to increased sensitivity to ionizing radiation and PARP1 inhibition. Lastly, POLA2 expression is elevated in glioblastoma multiforme tumors and correlates with poor overall patient survival. These data demonstrate a role for POLA2 in DSB repair and resistance to genotoxic stress.

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The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

FEMS Microbiology Reviews ◽

10.1093/femsre/fuaa009 ◽

2020 ◽

Vol 44 (3) ◽

pp. 351-368 ◽

Cited By ~ 2

Author(s):

Anurag Kumar Sinha ◽

Christophe Possoz ◽

David R F Leach

Keyword(s):

Dna Replication ◽

Cell Viability ◽

Strand Break ◽

Double Strand Break ◽

Replication Initiation ◽

Genome Replication ◽

Chromosomal Dna ◽

Dsb Repair ◽

Bacterial Dna ◽

Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

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The power of DNA double‐strand break (DSB) repair testing to predict breast cancer susceptibility

The FASEB Journal ◽

10.1096/fj.11-200790 ◽

2012 ◽

Vol 26 (5) ◽

pp. 2094-2104 ◽

Cited By ~ 24

Author(s):

Marlen Keimling ◽

Miriam Deniz ◽

Dominic Varga ◽

Andreea Stahl ◽

Hubert Schrezenmeier ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Susceptibility ◽

Strand Break ◽

Breast Cancer Susceptibility ◽

Double Strand Break ◽

Dsb Repair ◽

Dna Double Strand Break

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RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

Molecular and Cellular Biology ◽

10.1128/mcb.25.8.3127-3139.2005 ◽

2005 ◽

Vol 25 (8) ◽

pp. 3127-3139 ◽

Cited By ~ 108

Author(s):

Julie S. Martin ◽

Nicole Winkelmann ◽

Mark I. R. Petalcorin ◽

Michael J. McIlwraith ◽

Simon J. Boulton

Keyword(s):

Caenorhabditis Elegans ◽

Strand Break ◽

Double Strand Break ◽

Nonhomologous End Joining ◽

Related Protein ◽

End Joining ◽

Dsb Repair ◽

Phenotypic Differences ◽

Dna Double Strand Break ◽

Radiation Induced

ABSTRACT The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.

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