Hed1 regulates Rad51-mediated recombination via a novel mechanism

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination

Novel attributes of Hed1 affect dynamics and activity of the Rad51 presynaptic filament during meiotic recombination

During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase–double Holliday junction dissolvasome

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIα and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM–Topo IIIα–BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIα and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome

PGENETICS_D_13_01530R2_ReCombine_data

PGENETICS_D_13_01530R2_ReCombine_dat

Various phenotypes obtained from sequencing tetrads from various strains derived from the S288c/YJM789 background.

<p>^a Values indicate the standard deviation.</p><p>^b multicomparison Tukey test showing P values relative to wild type.</p><p>^c Z test of proportions.</p><p>^d The γ value is a measure of chromosome interference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004005#pgen.1004005-Chen1" target="_blank">[17]</a>.</p><p>^e Bootstrapping analysis, applied as described in Ref. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004005#pgen.1004005-Efron1" target="_blank">[93]</a>.</p

<i>S. cerevisiae</i> strains^a.

<p>^a Superscript “2” indicates that the plasmid was integrated into both haploid parents and the diploid is therefore homozygous.</p

Meiotic joint molecule analysis in various SK1 <i>dmc1-T159A ndt80</i>Δ strains.

<p><i>ndt80</i>Δ (NH2188), <i>hed1</i>Δ <i>ndt80</i>Δ <i>RAD54-T132A</i> (NH2223::pHN104(S/N)², <i>dmc1-T159A ndt80</i>Δ (NH2235), <i>dmc1-T159A hed1</i>Δ <i>ndt80</i>Δ (NH2190) and <i>dmc1-T159A hed1</i>Δ <i>ndt80</i>Δ <i>RAD54-T132A</i> (NH2193) diploids were transferred to Spo medium for nine hours to arrest the cells in pachytene and the DNA was crosslinked with psoralen, extracted and digested with XhoI. Color coding is the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004005#pgen-1004005-g002" target="_blank">Figure 2</a>. A. Southern blots of two-dimensional gels probed to detect interhomolog JMs (indicated by black arrows) and intersister JMs (indicated by red arrows) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004005#pgen.1004005-Oh1" target="_blank">[64]</a>. B. Quantitation of the ratio of interhomolog:intersister joint molecules in the gels shown in A averaged with a second replicate. Error bars indicate the standard error.</p

Two-hybrid interactions between <i>DMC1</i> and <i>dmc1-T159A</i>.

<p>^a Two duplicate samples were assayed for three independent transformants. The values for each transformant were then averaged and the standard deviation calculated. β-galactosidase activity is indicated in Miller units <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004005#pgen.1004005-Miller1" target="_blank">[89]</a>.</p

Biochemical characterization of recombinant Dmc1-T159A protein.

<p>A. For Dmc1 and Dmc1-T159A, 2.5 µg of each protein were analyzed by SDS-polyacrylimide gel electrophoresis (PAGE) and Coomassie staining. B. The stability of Dmc1 filaments on bead-immobilized ssDNA was assessed by exposing the filaments to RPA and measuring the amount of Dmc1 retained on the beads (n = 3, +/− standard error). Proteins were monitored by SDS-PAGE and Coomassie staining. The experiments were performed with different Ca²⁺ concentrations as indicated. Rfa2, the 30 kDa subunit of RPA is indicated. The histogram shows the percent of Dmc1 protein that remained associated with the beads after challenge by RPA as determined by band desitometry. C. (i) Schematic of the strand exchange recombination assay. (ii) Strand exchange activity of Dmc1 and Dmc1-T159A was monitored using 2, 4, or 8 µM protein in the presence of either 10 µM or 1 mM Ca²⁺. The histogram indicates the percent of radioactively labeled oligonucleotide that was incorporated into the slower migrating product by strand exchange (n = 3, +/− standard error). At 1 mM Ca²⁺, the inhibition of strand exchange seen at elevated concentrations of wild-type Dmc1 is likely due to coating of the dsDNA substrate by the recombinase, thereby blocking access of the ssDNA filament. D. Dmc1 and Dmc1-T159A interactions with Rad54 and Rdh54 were assayed by pull-down experiments. S-tagged Rad54 or Rdh54 (2 µg each) was incubated with 1.2 µg Dmc1 or Dmc1-T159A. The S-tagged protein was captured on S-protein agarose resin, which was washed and the protein eluted with SDS. S = supernatant after collecting the beads, W = supernatant obtained from washing the beads, E = eluate from beads. The “-” indicates that no tagged protein was added to the reaction. E. (i) Schematic of the D-loop recombination assay. (ii) Homologous DNA pairing activity of Dmc1 and Dmc1-T159A was assessed by a D-loop formation assay (n = 3, +/− standard error). 0.5 or 1.0 µM of Dmc1 or Dmc1-T159A was combined with 0, 150, or 250 nM of Rdh54. The histogram shows the percent of radioactive ssDNA that is incorporated into the slower migrating D-loop product via homologous pairing.</p