Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Xu, Jingfei; Zhao, Lingyun; Peng, Sijia; Chu, Huiying; Liang, Rui; Tian, Meng; Connell, Philip P.; Li, Guohui; Chen, Chunlai; Wang, Hongwei

doi:10.1093/nar/gkab1141

“…To investigate whether DMC1 inhibits RAD51, we took the approach of ectopically expressing DMC1 in somatic cells in which normally only RAD51 is present. RAD51 and DMC1 share similar biochemical properties and catalyze essentially the same DNA strand exchange reaction [16,26,33,34,67], although DMC1 fibres are able to accommodate unpaired base mismatches [88][89][90][91][92] and results from fission yeast indicate opposite polarities of nucleofilament polymerization of the two proteins [16,93]. It is not yet clear whether functional differences between RAD51 and DMC1 result from distinct biochemical properties or influence of different co-factors.…”

Section: Replacement Of Rad51 In Somatic Cells With Dmc1: Mitotic Hr ...mentioning

confidence: 99%

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Ines

¹

,

Bazile

²

,

Gallego

³

et al. 2022

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Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.

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“…Structural differences between Rad51 and Dmc1 that might reflect a change in mismatch tolerance have also been reported (54,55).…”

Section: Introductionmentioning

confidence: 71%

“…Such an increased tolerance has been suggested by in vitro studies of DNA strand exchange involving short oligonucleotides, in which Dmc1, but not Rad51, could “step over” a mismatch in the formation of a displacement loop (D-loop) intermediate (51–53). Structural differences between Rad51 and Dmc1 that might reflect a change in mismatch tolerance have also been reported (54, 55).…”

Section: Introductionmentioning

confidence: 78%

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

Choi

¹

,

Xue

²

,

Cao

³

et al. 2022

Preprint

0

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In many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51’s homolog, Dmc1, acting with Rad54’s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease, either under control of a galactose-inducible promoter or a meiosis-specific SPO13 promoter. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which a 108-bp homologous sequence shared between the DSB end and the donor sequence could be easily modified. As previously shown for a haploid mitotic strain, BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic haploids or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Rad51 took over from Dmc1 in both hed1Δ and dmc1Δ hed1Δ mutants. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination in budding yeast. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. In all cases, assimilation of mismatches into the BIR product was dependent on the 3’ to 5’ exonuclease activity of DNA polymerase δ.Author SummaryIn many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In budding yeast meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51’s homolog, Dmc1, acting with Rad54’s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which recombination occurred between a 108-bp homologous sequence shared between the DSB end and the donor sequence. The donor sequence could be easily modified to introduce different arrangements of mismatches. BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Dmc1 was deleted and Rad51 was activated. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. As in mitotic cells, the assimilation of mismatches into the BIR product was dependent on the 3’ to 5’ exonuclease activity of DNA polymerase δ.

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“…The copyright holder for this preprint (which this version posted July 5, 2022. ; https://doi.org/10.1101/2022.07.05.498790 doi: bioRxiv preprint unpaired base mismatches [88][89][90][91][92] and results from fission yeast indicate opposite polarities of nucleofilament polymerization of the two proteins [16,93]. It is not yet clear whether functional differences between RAD51 and DMC1 result from distinct biochemical properties or influence of different co-factors.…”

Section: Replacement Of Rad51 In Somatic Cells With Dmc1 : Mitotic Hr...mentioning

confidence: 99%

“…To investigate whether DMC1 inhibits RAD51, we took the approach of ectopically expressing DMC1 in somatic cells in which normally only RAD51 is present. RAD51 and DMC1 share similar biochemical properties and catalyze essentially the same DNA strand exchange reaction [16,26,33,34,67], although DMC1 fibres are able to accommodate unpaired base mismatches [88][89][90][91][92] and results from fission yeast indicate opposite polarities of nucleofilament polymerization of the two proteins [16,93]. It is not yet clear whether functional differences between RAD51 and DMC1 result from distinct biochemical properties or influence of different co-factors.…”

Section: Replacement Of Rad51 In Somatic Cells With Dmc1 : Mitotic Hr...mentioning

confidence: 99%

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Ines

¹

,

Bazile

²

,

Gallego

³

et al. 2022

Preprint

0

View full text Add to dashboard Cite

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.Author SummaryEssential for fertility and responsible for a major part of genetic variation in sexually reproducing species, meiotic recombination establishes the physical linkages between homologous chromosomes which ensure their balanced segregation in the production of gametes. These linkages, or chiasmata, result from DNA strand exchange catalyzed by the RAD51 and DMC1 recombinases and their numbers and distribution are tightly regulated. Essential for maintaining chromosomal integrity in mitotic cells, the strand-exchange activity of RAD51 is downregulated in meiosis, where it plays a supporting role to the activity of DMC1. Notwithstanding considerable attention from the genetics community, precisely why this is done and the mechanisms involved are far from being fully understood. We show here in the plant Arabidopsis that DMC1 can downregulate RAD51 strand-exchange activity and propose that this may be a general mechanism for suppression of RAD51-mediated recombination in meiosis.

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Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Cited by 29 publications

References 71 publications

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

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