PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

AbstractStalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

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PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

10.1101/773242 ◽

2019 ◽

Cited By ~ 3

Author(s):

Ann Liza Piberger ◽

Akhil Bowry ◽

Richard D W Kelly ◽

Alexandra K Walker ◽

Daniel Gonzalez ◽

...

Keyword(s):

Homologous Recombination ◽

Dna Adducts ◽

Mammalian Cells ◽

Genetic Recombination ◽

Sister Chromatid Exchanges ◽

Environmental Carcinogens ◽

Gap Formation ◽

Bulky Dna Adducts ◽

Fork Stalling ◽

Dna Template

AbstractObstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and other bulky model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

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The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation

10.1101/2021.05.27.446004 ◽

2021 ◽

Author(s):

Laurent G Maloisel ◽

Emilie Ma ◽

Eric Coic

Keyword(s):

Homologous Recombination ◽

Uv Radiation ◽

Dna Lesions ◽

Template Switching ◽

Dna Double Strand Breaks ◽

Dna Damage Tolerance ◽

Strand Breaks ◽

Rad51 Paralogs ◽

Replicative Polymerases ◽

Stalled Replication Forks

Bypass of DNA lesions that block replicative polymerases during DNA replication relies on several DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway involves specialized DNA polymerases that incorporate nucleotides in front of base lesions. The template switching and the homologous recombination (HR) pathways are mostly error-free because the bypass is performed by using typically the sister chromatid as a template. This is promoted by the Rad51 recombinase that forms nucleoprotein filaments on single-strand DNA (ssDNA). The balance between error-prone and error-free pathways controls the level of mutagenesis. In yeast, the Rad55-Rad57 complex of Rad51 paralogs is required for Rad51 filament formation and stability, notably by counteracting the Srs2 antirecombinase. Several reports showed that Rad55-Rad57 promotes HR at stalled replication forks more than at DNA double-strand breaks (DSB), suggesting that this complex is more efficient at ssDNA gaps and thus, could control the recruitment of TLS polymerases. To address this point, we studied the interplay between Rad55-Rad57 and the TLS polymerases Polζ and Polη following UV radiation. We confirmed that Rad55-Rad57 protects Rad51 filaments from Srs2 dismantling activity but we found that it is also essential for the promotion of UV-induced HR independently of Srs2. In addition, we observed that cell UV sensitivity, but not DSB sensitivity, is synergistically increased when Rad55 and Polζ deletions are combined. Moreover, we found that mutagenesis and HR frequency were increased in rad55∆ mutants and in TLS-deficient cells, respectively. Finally, UV-induced HR was partially restored in Rad55-deficient cells with mutated Polζ or Polη. Overall, our data suggest that the HR and TLS pathways compete for the same ssDNA substrates and that the Rad55-Rad57 complex of Rad51 paralogs prevents the recruitment of TLS polymerases and counterbalances mutagenesis.

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Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3331-3336.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3331-3336

Author(s):

K Y Song ◽

L Chekuri ◽

S Rauth ◽

S Ehrlich ◽

R Kucherlapati

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Recombination Frequency ◽

Double Strand Breaks ◽

Base Pairs ◽

In Vitro System ◽

Strand Breaks ◽

Cell Extracts ◽

Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

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Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6386 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6386-6393 ◽

Cited By ~ 114

Author(s):

D G Taghian ◽

J A Nickoloff

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Mammalian Cells ◽

Chinese Hamster ◽

Double Strand Breaks ◽

Chromosomal Dna ◽

Exogenous Dna ◽

Strand Breaks ◽

Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

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Double-strand-break-induced homologous recombination in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst0290196 ◽

2001 ◽

Vol 29 (2) ◽

pp. 196-201 ◽

Cited By ~ 186

Author(s):

R. D. Johnson ◽

M. Jasin

Keyword(s):

Homologous Recombination ◽

Sister Chromatid ◽

Mammalian Cells ◽

Indirect Evidence ◽

Strand Break ◽

Double Strand Breaks ◽

Crossing Over ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

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Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

International Journal of Hyperthermia ◽

10.1080/02656736.2016.1252989 ◽

2016 ◽

Vol 33 (3) ◽

pp. 336-342 ◽

Cited By ~ 6

Author(s):

Akihisa Takahashi ◽

Eiichiro Mori ◽

Yosuke Nakagawa ◽

Atsuhisa Kajihara ◽

Tadaaki Kirita ◽

...

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.267 ◽

1997 ◽

Vol 17 (1) ◽

pp. 267-277 ◽

Cited By ~ 176

Author(s):

R G Sargent ◽

M A Brenneman ◽

J H Wilson

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Repair Process ◽

Strand Break ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Site Specific ◽

Strand Breaks

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

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Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

Nature Communications ◽

10.1038/ncomms12889 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 56

Author(s):

Pauline Chanut ◽

Sébastien Britton ◽

Julia Coates ◽

Stephen P. Jackson ◽

Patrick Calsou

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Exonuclease Activity ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Additional Mechanism ◽

Strand Breaks ◽

Rad51 Focus ◽

Dna Resection

Abstract Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.

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Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates

Nucleic Acids Research ◽

10.1093/nar/gkz1077 ◽

2019 ◽

Vol 48 (3) ◽

pp. e13-e13 ◽

Cited By ~ 5

Author(s):

Le P Ngo ◽

Norah A Owiti ◽

Carol Swartz ◽

John Winters ◽

Yang Su ◽

...

Keyword(s):

Comet Assay ◽

Dna Adducts ◽

Excision Repair ◽

Environmental Chemicals ◽

Single Strand ◽

Strand Breaks ◽

Single Strand Breaks ◽

Bulky Dna Adducts ◽

Dna Strand

Abstract Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

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