scholarly journals DNA polymerase theta suppresses mitotic crossing over

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009267
Author(s):  
Juan Carvajal-Garcia ◽  
K. Nicole Crown ◽  
Dale A. Ramsden ◽  
Jeff Sekelsky
Keyword(s):  
Homologous Recombination ◽  
Loss Of Heterozygosity ◽  
Mammalian Cells ◽  
Holliday Junction ◽  
Crossing Over ◽  
Chromosome Break ◽  
End Joining ◽  
Dna Damaging Agents ◽  
Junction Formation ◽  
Dna Substrate

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

scholarly journals DNA polymerase theta suppresses mitotic crossing over

2020 ◽  
Author(s):  
Juan Carvajal-Garcia ◽  
K. Nicole Crown ◽  
Dale A. Ramsden ◽  
Jeff Sekelsky
Keyword(s):  
Homologous Recombination ◽  
Loss Of Heterozygosity ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Holliday Junction ◽  
Crossing Over ◽  
Chromosome Break ◽  
Repair Pathway ◽  
Chromosome Breaks ◽  
Break Repair

AbstractPolymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol θ to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

scholarly journals Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways

2020 ◽  
Vol 295 (37) ◽  
pp. 12946-12961
Author(s):  
Soichiro S. Ito ◽  
Yosuke Nakagawa ◽  
Masaya Matsubayashi ◽  
Yoshihiko M. Sakaguchi ◽  
Shinko Kobashigawa ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Checkpoint ◽  
Nonhomologous End Joining ◽  
Homologous Recombination Repair ◽  
Dna Breaks ◽  
End Joining ◽  
Nucleotide Synthesis ◽  
Recombination Repair ◽  
Repair Pathways

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV– and chemotherapeutic drug–induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU–treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU–based chemotherapy.

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

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
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.

scholarly journals RAD51 Is Required for the Repair of Plasmid Double-Stranded DNA Gaps from Either Plasmid or Chromosomal Templates

2000 ◽  
Vol 20 (4) ◽  
pp. 1194-1205 ◽  
Author(s):  
Stephan Bärtsch ◽  
Leslie E. Kang ◽  
Lorraine S. Symington
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Radiation Chemical ◽  
Strand Breaks ◽  
Independent Events ◽  
In The Wild ◽  
A Minor

ABSTRACT DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52epistasis group was tested in this system. RAD51,RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered fromrad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, orRAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not onRAD51. The residual repair events in rad51mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms forRAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically inrad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.

scholarly journals Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

2001 ◽  
Vol 21 (10) ◽  
pp. 3425-3435 ◽  
Author(s):  
Mark D. Baker ◽  
Erin C. Birmingham
Keyword(s):  
Homologous Recombination ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Holliday Junction ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Break Repair

ABSTRACT In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

scholarly journals Fission Yeast Mus81·Eme1 Holliday Junction Resolvase Is Required for Meiotic Crossing Over but Not for Gene Conversion

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 2289-2293 ◽  
Author(s):  
Gerald R Smith ◽  
Michael N Boddy ◽  
Paul Shanahan ◽  
Paul Russell
Keyword(s):  
Homologous Recombination ◽  
Fission Yeast ◽  
Gene Conversion ◽  
Protein Complex ◽  
Holliday Junctions ◽  
Holliday Junction ◽  
Crossing Over ◽  
Dna Structures ◽  
Branched Dna ◽  
Holliday Junction Resolvase

Abstract Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81·Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81·Eme1 protein complex resolves Holliday junctions in meiotic cells.

Sign in / Sign up

Export Citation Format

Share Document