Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

Kaniecki, Kyle; Tullio, Luisina De; Gibb, Bryan; Kwon, Youngho; Sung, Patrick; Greene, Eric C.

doi:10.1016/j.celrep.2017.11.047

Cited by 46 publications

(129 citation statements)

References 47 publications

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“…In these assays, unlabeled Srs2 disrupts Rad51-ssDNA, allowing for the rapid replacement of Rad51 by RPA-GFP, and single Srs2 complexes can translocate over distances spanning thousands of nucleotides ( Fig. 1D) (44). In striking contrast, we found no evidence of Srs2 translocation on Dmc1-ssDNA even in assays containing fivefold more Srs2 (500 pM) relative to typical assays with Rad51-ssDNA ( Fig.…”

Section: Resultsmentioning

confidence: 74%

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Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Crickard

Kaniecki

Kwon

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesisdependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosisspecific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

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Section: Resultsmentioning

confidence: 74%

“…We have established ssDNA curtain assays for visualizing the behaviors of Srs2 on HR intermediates in real time (Fig. 1C) (42,44,45). In brief, long ssDNA substrates (≥50 kilonucleotides) are generated by rolling circle replication using a 5′ biotinylated primer (45)(46)(47).…”

Section: Resultsmentioning

confidence: 99%

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Crickard

Kaniecki

Kwon

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…For all two-color images, we use a custom-built shuttering system to avoid the bleed through from the green into the red channel during image acquisition. With this system, images from the green (GFP) and the red (mCherry) channels are recorded independently, these recordings are offset by 100 milliseconds such that when one camera records the red channel image, the green laser is shuttered off, and vice versa (30)(31)(32).…”

Section: Methodsmentioning

confidence: 99%

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

Crickard

Kaniecki

Kwon

et al. 2018

Journal of Biological Chemistry

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During meiosis, the two DNA recombinases Rad51 and Dmc1, form specialized presynaptic filaments that are adapted for performing recombination between homologous chromosomes. There is currently a limited understanding of how these two recombinases are organized within the meiotic presynaptic filament. Here, we used single molecule imaging to examine the properties of presynaptic complexes comprised of both Rad51 and Dmc1. We demonstrate that Rad51 and Dmc1 have an intrinsic ability to selfsegregate, even in the absence of any other recombination accessory proteins. Moreover, we found that the presence of Dmc1 stabilizes the adjacent Rad51 filaments, suggesting that crosstalk between these two recombinases may affect their biochemical properties. Based upon these findings, we describe a model for the organization of Rad51 and Dmc1 within the meiotic presynaptic complex, which is also consistent with in vivo observations, genetic findings and biochemical expectations. This model argues against the existence of extensively intermixed filaments, and we propose that Rad51 and Dmc1 have intrinsic capacities to form spatially distinct filaments, suggesting that additional recombination cofactors are not required to segregate the Rad51 and Dmc1 filaments.

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“…Rad51 is also negatively regulated by the budding yeast Srs2 protein, a 3´ to 5´ DNA helicase/translocase of the UvrD family (reviewed in Niu and Klein, 2017). Srs2 interacts with Rad51 filaments in vitro and strips them from DNA (Kaniecki et al, 2017;Krejci et al, 2003;Veaute et al, 2003). Loss of Srs2 activity in mitotic cells leads to DNA damage sensitivity, genome instability, reduced DSB repair efficiency, and an increase in COs among repair products (Elango et al, 2017;Ira et al, 2003;Lawrence and Christensen, 1979;Marini and Krejci, 2010;Rong et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

“…As srs2 sensitivity to DNA damage is partially suppressed by deletion of RAD51, it is thought that srs2 mutant phenotypes relate to failures in Rad51 removal from ssDNA (Ira et al, 2003;Krejci et al, 2003). Srs2 also unwinds branched DNA structures in vitro, including those mimicking D-loop recombination intermediates, consistent with a role in promoting SDSA (Dupaigne et al, 2008;Kaniecki et al, 2017;Liu et al, 2017;Marini and Krejci, 2012). However, this function has yet to be fully investigated in vivo.…”

Section: Introductionmentioning

confidence: 99%

S. cerevisiaeSrs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

Hunt

Ahmed

Kaur

et al. 2019

Preprint

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We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilizes Rad51-DNA filaments, and is thought to regulate strand invasion and prevent hyperrecombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalized with the ssDNAbinding protein RPA, suggesting the presence of persistent singlestrand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity, and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards inter-homologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

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Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

Cited by 46 publications

References 47 publications

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

S. cerevisiaeSrs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

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