scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

Abstract Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

AbstractMeiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

scholarly journals Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yann Duroc ◽  
Rajeev Kumar ◽  
Lepakshi Ranjha ◽  
Céline Adam ◽  
Raphaël Guérois ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Recombination Hotspots ◽  
Genome Wide ◽  
D Loop ◽  
Mismatch Recognition ◽  
Gene Conversion Tract ◽  
Gene Conversions ◽  
Conversion Tract

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

2-Nitropropane induces DNA repair synthesis in rat hepatocytes in vitro and in vivo

1988 ◽  
Vol 9 (5) ◽  
pp. 811-815 ◽  
Author(s):  
U. Andrae ◽  
L. Vogl ◽  
J. Lichtmannegger ◽  
K.H. Summer
Keyword(s):  
Dna Repair ◽  
Rat Hepatocytes ◽  
Repair Synthesis ◽  
Dna Repair Synthesis

scholarly journals Rad52 oligomeric N-terminal domain stabilizes Rad51 nucleoprotein filaments and contributes to their protection against Srs2

2021 ◽  
Author(s):  
Emilie Ma ◽  
Laurent Maloisel ◽  
Lea Le Falher ◽  
Raphael Guerois ◽  
Eric Coic
Keyword(s):  
Genome Stability ◽  
Ring Structure ◽  
Homologous Sequence ◽  
Filament Formation ◽  
Repair Synthesis ◽  
Terminal Domain ◽  
Mediator Protein ◽  
Dna Repair Synthesis

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells without disturbing Rad52 mediator and pairing activity, both in vivo and in vitro. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, our findings indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

scholarly journals Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

2013 ◽  
Vol 42 (4) ◽  
pp. 2380-2390 ◽  
Author(s):  
Shreya Paliwal ◽  
Radhakrishnan Kanagaraj ◽  
Andreas Sturzenegger ◽  
Kamila Burdova ◽  
Pavel Janscak
Keyword(s):  
Molecular Mechanisms ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Inhibitory Effect ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Break Site ◽  
Strand Annealing

Abstract Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.

scholarly journals Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.

1995 ◽  
Vol 15 (4) ◽  
pp. 1993-1998 ◽  
Author(s):  
L Li ◽  
C A Peterson ◽  
X Lu ◽  
R J Legerski
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Repair Synthesis ◽  
Damage Site ◽  
Cell Extracts ◽  
Nucleotide Excision ◽  
Delta G ◽  
Dna Repair Synthesis

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

scholarly journals Differential Regulation of Short- and Long-Tract Gene Conversion between Sister Chromatids by Rad51C

2006 ◽  
Vol 26 (21) ◽  
pp. 8075-8086 ◽  
Author(s):  
Ganesh Nagaraju ◽  
Shobu Odate ◽  
Anyong Xie ◽  
Ralph Scully
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Sister Chromatids ◽  
Almost All ◽  
A Site ◽  
Gene Conversion Tract ◽  
Gene Conversions

ABSTRACT The Rad51 paralog Rad51C has been implicated in the control of homologous recombination. To study the role of Rad51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster Rad51C −/− CL-V4B cells in response to a site-specific chromosomal double-strand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type Rad51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type Rad51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking Rad51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that Rad51C plays a pivotal role in determining the “choice” between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination.

scholarly journals Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1467
Author(s):  
Emilie Ma ◽  
Laurent Maloisel ◽  
Léa Le Falher ◽  
Raphaël Guérois ◽  
Eric Coïc
Keyword(s):  
Genome Stability ◽  
Ring Structure ◽  
Homologous Sequence ◽  
Filament Formation ◽  
Repair Synthesis ◽  
Terminal Domain ◽  
Mediator Protein ◽  
Dna Repair Synthesis

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52–Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

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