Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jan 1;23(1):67-79.
doi: 10.1101/gad.1737809.

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Rohit Prakash  1 , Dominik Satory, Eloïse Dray, Almas Papusha, Jürgen Scheller, Wilfried Kramer, Lumir Krejci, Hannah Klein, James E Haber, Patrick Sung, Grzegorz Ira
Affiliations

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Rohit Prakash et al. Genes Dev. .

Abstract

Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Screen for genes regulating the crossover frequency identifies Mph1. (A) Ectopic gene conversion is induced by DSBs generated by the HO endonuclease within a 1.9-kb MAT a sequence (gray rectangle) that replaced the ARG5,6 gene on chromosome V. The MAT a-inc sequence on chromosome III is a donor for recombination, and shares 1403-bp and 530-bp homology on opposite sides of the DSB. EcoRI noncrossover fragments (NCOs) of 6.4 kb and 3 kb can be distinguished from crossover fragments (COs) of 6 kb and 3.4 kb, and quantified on Southern blots. The probe used to detect crossovers and noncrossovers was a MAT a fragment overlapping the first 200 bp on each side of the HO break. (B) Viability resulting from DSB repaired by ectopic recombination was measured by dividing colony-forming units on YEP-galactose over those on YEP-dextrose. Crossover frequency among the product in SRS2 synthetic interactors was determined 8 h after break induction as the intensity ratio of the Southern blot signal corresponding to gene conversion with crossover and that corresponding to gene conversion both with and without crossover.
Figure 2.
Figure 2.
MPH1 helicase channels DSB repair to noncrossovers. (A) Kinetics of DSB repair in wild type and mph1Δ mutant, determined by dividing the normalized Southern blot signals corresponding to product at different times by the signal corresponding to the maximal product at 8 h after break induction in wild-type cells. (B) Comparison of growth rate between srs2Δ and mph1Δ. Slow growth rate of double mutant srs2Δ mph1Δ is suppressed by elimination of RAD51. (C) Crossover level among products in mph1Δ and helicase point mutants is shown. The mph1Δ strain was complemented with plasmids carrying either wild-type or mutant MPH1 sequences.
Figure 3.
Figure 3.
Mph1 is recruited to DSBs, and works independently of Sgs1 and Srs2. (A, panel I) Southern blot analysis of gene conversion with and without crossovers in strains lacking the indicated helicases. (Panel II, top) Percentage of crossovers in cells that repaired the DSB. (Bottom) Percentage of crossovers (black) and noncrossovers (gray) among all cells with the HO cut. These percentages were determined by dividing the normalized intensities of the crossover or noncrossover band on Southern blot by the intensity of parental uncut MATa band before break induction (time, 0 h) (B) Time-course ChIP experiments showing recruitment of TAP-tagged Mph1 to nonrepairable HO break (strain JKM179) or to the HMLα donor sequence in strain (JKM 161) that can repair the break by gene conversion.
Figure 4.
Figure 4.
DNA binding and D-loop unwinding by Mph1. (A) DNA substrates used for DNA-binding and DNA-unwinding assays. The oligonucleotides used for constructing the substrates and the sizes of the DNA regions in the substrates are indicated and their sequences are given in Supplemental Table 2. In the central portion of the D-loop substrates, the unpaired strand bears no homology with the paired duplex region. (B) Mph1 (10–200 nM) was incubated with the 5′ D-loop substrate (50 nM) and dsDNA (50 nM) (panel I) or ssDNA (50 nM) (panel II). The results from these DNA mobility shift experiments were plotted. (C) Mph1 (5 to 40 nM) was incubated with D-loop substrates (50 nM each) that harbored a 5′ tail (panel I), a 3′ tail (panel II), or no tail (panel III). The heat-denatured substrate (HD) was run in lane 1 and the reaction blank was run in lane 2. The results from these DNA unwinding experiments were plotted in panel IV.
Figure 5.
Figure 5.
Effect of Mph1 on the Rad51-mediated D-loop reaction. (A) Schematic of the D-loop reaction. The ssDNA substrate was the 90-mer Oligonucleotide D1, homologous to positions 1932–2022 of pBluescript SK DNA (see Supplemental Table 2 for detailed sequence). (B) D-loop reactions without (lane 2) or with (lanes 3–6) Mph1 (50, 100, 150, and 200 nM) were analyzed after 4 or 8 min of incubation. The reaction blank (Bl) was run in lane 1. The results were plotted. (C) D-loop reactions without (lane 2) or with (lanes 3–6) mph1 D209N (50, 100, 150, and 200 nM) were analyzed after 8 min of incubation. The reaction blank (Bl) was run in lane 1. The results were plotted. The concentration of Rad51 was 0.8 μM and of Oligonucleotide D1 was 2.4 μM (nucleotides).
Figure 6.
Figure 6.
Mph1 acts by dissociating preformed D-loops. (A) Summary of D-loop reactions that did not contain Mph1 (i) or with Mph1 (50, 100, 200 nM) added at different stages (ii–iv). The reactions used Oligonucleotide D1 as the ssDNA substrate. (B) Results from the reactions summarized in A. The reaction blank was run in lane 1. The results were plotted. (C) Schematic of D-loop reactions utilizing ssDNA substrates that gave no tail (90-mer Oligonucleotide D1), a 50-nucleotide 3′ tail (the 140-mer Oligonucleotide D2), or a 50-nucleotide 5′ tail (the 140-mer Oligonucleotide D3). All three oligonucleotides bear homology with positions 1932 to 2022 of pBluescript SK DNA and their sequence is given in Supplemental Table 2. (D–F) Time course experiments that examined formation the D-loops with no tail (D), a 3′ 50-nucleotide tail (E), or a 5′ 50-nucleotide tail (F). These reactions either contained Mph1 (150 nM; lanes 8–13) or not (lanes 2–7). In D, 2.4 μM (nucleotides) of Oligonucleotide D1 and 0.8 μM of Rad51 were used; in E and F, 3.72 μM of Oligonucleotide D2 or D3 and 1.24 μM of Rad51 were used. The reaction blank (Bl) was run in lane 1. The results were plotted.
Figure 7.
Figure 7.
Mechanism and specificity of Mph1 action. (A) In I, Mph1 or Srs2 (50 or 100 nM) was added with Rad51 at the beginning of the D-loop reaction, which, following the incorporation of pBluescript SK DNA, was incubated for 8 min. The reaction blank and the reaction without any helicase were run in lanes 1 and 2, respectively. The results were plotted. In II, Mph1 or Srs2 (50 or 100 nM) was added to the D-loop reaction 1 min after product synthesis had commenced, followed by an additional 8-min incubation. The reaction blank and the reaction without any helicase were run in lanes 1 and 2, respectively. The results were plotted. Oligonucleotide D1 was used at 2.4 μM (nucleotides) and Rad51 was at 0.8 μM, as in Figure 6. (B) Mph1, BLM, RecQ1, and WRN (50 or 100 nM) were tested for their ability to dissociate human Rad51-made D-loops that harbored no tail (I), a 3′ tail (II), or a 5′ tail (III). The D-loops were generated with Oligonucleotide D1 (2.4 μM nucleotides with 0.8 μM Rad51), D2 (3.72 μM nucleotides with 1.24 μM Rad51), or D3 (3.73 μM nucleotides with 1.24 μM Rad51) and pBluescript SK duplex DNA, as in Figure 6. The helicases were added with Rad51 at the beginning of the reaction and the incubation time was 8 min. The reaction blank and the reaction without any helicase were run in lanes 1 and 2, respectively. The results were plotted.
Figure 8.
Figure 8.
Model depicting regulation of exchange frequency by DNA helicases during DSB-induced recombination. (A) Both Mph1 and Srs2 promote the noncrossover SDSA pathway by minimizing the possibility of creating a dHJ. Mph1 displaces the invading strand after DNA synthesis has commenced, thus preventing dHJ formation and promoting SDSA. Mph1 may also inhibit recombination if it dissociates D-loop before invading strand-initiated DNA synthesis. Srs2 possibly removes Rad51 from the unpaired 3′ DNA tail to prevent second end capture and dHJ formation. (B) When the D-loop becomes extended and more stable, dHJs are formed and can be resolved into crossovers and noncrossovers by a yet-unknown resolvase. (C) Sgs1 helicase in complex with Rmi1 and Top3 resolves these mitotic dHJs into noncrossovers.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allers T., Lichten M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 2001;106:47–57. - PubMed
    1. Barbera M.A., Petes T.D. Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae . Proc. Natl. Acad. Sci. 2006;103:12819–12824. - PMC - PubMed
    1. Beumer K.J., Pimpinelli S., Golic K.G. Induced chromosomal exchange directs the segregation of recombinant chromatids in mitosis of Drosophila . Genetics. 1998;150:173–188. - PMC - PubMed
    1. Bridge W.L., Vandenberg C.J., Franklin R.J., Hiom K. The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair. Nat. Genet. 2005;37:953–957. - PubMed
    1. Brosh R.M., Jr, Orren D.K., Nehlin J.O., Ravn P.H., Kenny M.K., Machwe A., Bohr V.A. Functional and physical interaction between WRN helicase and human replication protein A. J. Biol. Chem. 1999;274:18341–18350. - PubMed

Publication types

MeSH terms