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

2019 ◽  
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.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ann Liza Piberger ◽  
Akhil Bowry ◽  
Richard D. W. Kelly ◽  
Alexandra K. Walker ◽  
Daniel González-Acosta ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Dna Adducts ◽  
Mammalian Cells ◽  
Genetic Recombination ◽  
Gap Formation ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Bulky Dna Adducts ◽  
Fork Stalling ◽  
Stalled Replication Forks

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.

scholarly journals The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

2004 ◽  
Vol 24 (13) ◽  
pp. 5776-5787 ◽  
Author(s):  
Laura J. Niedernhofer ◽  
Hanny Odijk ◽  
Magda Budzowska ◽  
Ellen van Drunen ◽  
Alex Maas ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Wild Type ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Normal Metabolism ◽  
Cross Links

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1 −/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

scholarly journals Detection of Aromatic DNA Adducts in Human Cells and Fish Liver

1989 ◽  
Vol 8 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Bruce P. Dunn
Keyword(s):  
Dna Adducts ◽  
High Performance ◽  
High Rate ◽  
Control Fish ◽  
Environmental Carcinogens ◽  
Mucosal Cells ◽  
Polycyclic Aromatic ◽  
Bulky Dna Adducts ◽  
Former Smokers ◽  
Layer Chromatography

Both nuclease PI treatment and reverse-phase high-performance liquid chromatography (HPLC) were used to enrich hydrophobic/bulky DNA adducts in DNA digests. 32P-postlabeling procedures and thin layer chromatography were then used to detect and quantitate aromatic/bulky DNA adducts. For both human and fish DNA from individuals exposed to environmental carcinogens, the nuclease PI and HPLC enrichment procedures generally gave similar results. The bottom sediments of the Buffalo and Detroit rivers are contaminated with polycyclic aromatic hydrocarbon carcinogens, and brown bullheads in these rivers show a high rate of liver cancer. Compared to DNA from control fish raised in aquariums, DNA of livers of brown bullheads from the polluted rivers exhibited elevated levels of DNA adducts. DNA from human oral mucosal cells and lymphocytes exhibited DNA adducts, but adducts levels did not differ significantly in smokers and nonsmokers. Adduct levels in DNA from human lung biopsy tissue, however, were elevated in smokers compared to nonsmokers. In former smokers, adducts levels were highest in those who had recently quit, and lowest in those who had not smoked for 10 years or more. Measurement of DNA adducts by 32P-postlabeling appears to be a useful and particularly direct procedure for assessing genetic damage from environmental carcinogens.

scholarly journals ATRX and RECQ5 define distinct homologous recombination subpathways

2021 ◽  
Vol 118 (3) ◽  
pp. e2010370118
Author(s):  
Amira Elbakry ◽  
Szilvia Juhász ◽  
Ki Choi Chan ◽  
Markus Löbrich
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Holliday Junctions ◽  
Nuclear Antigen ◽  
Sequence Information ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Strand Annealing

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

scholarly journals The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination

2019 ◽  
Vol 47 (19) ◽  
pp. 10151-10165 ◽  
Author(s):  
Julieta Martino ◽  
Gregory J Brunette ◽  
Jonathan Barroso-González ◽  
Tatiana N Moiseeva ◽  
Chelsea M Smith ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Complex Function ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Complex Functions ◽  
Replication Fork Stalling ◽  
Fork Stalling ◽  
Chromatid Exchanges

AbstractRAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

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