Disparate roles for C. elegans DNA translocase paralogs RAD-54.L and RAD-54.B in meiotic prophase germ cells

Abstract RAD54 family DNA translocases partner with RAD51 recombinases to ensure stable genome inheritance, exhibiting biochemical activities both in promoting recombinase removal and in stabilizing recombinase association with DNA. Understanding how such disparate activities of RAD54 paralogs align with their biological roles is an ongoing challenge. Here we investigate the in vivo functions of Caenorhabditis elegans RAD54 paralogs RAD-54.L and RAD-54.B during meiotic prophase, revealing distinct contributions to the dynamics of RAD-51 association with DNA and to the progression of meiotic double-strand break repair (DSBR). While RAD-54.L is essential for RAD-51 removal from meiotic DSBR sites to enable recombination progression, RAD-54.B is largely dispensable for meiotic DSBR. However, RAD-54.B is required to prevent hyperaccumulation of RAD-51 on unbroken DNA during the meiotic sub-stage when DSBs and early recombination intermediates form. Moreover, DSB-independent hyperaccumulation of RAD-51 foci in the absence of RAD-54.B is RAD-54.L-dependent, revealing a hidden activity of RAD-54.L in promoting promiscuous RAD-51 association that is antagonized by RAD-54.B. We propose a model wherein a division of labor among RAD-54 paralogs allows germ cells to ramp up their capacity for efficient homologous recombination that is crucial to successful meiosis while counteracting potentially deleterious effects of unproductive RAD-51 association with unbroken DNA.


Meiotic crossover (CO) distribution assay using SNP mapping strategy
Meiotic CO distribution was assayed as described (9).TG3319(rad-54.B(gk340656)) was crossed to CB4856 to generate a new strain XSW933 homozygous for the rad-54B(gk340656) mutation as well as the CB4856-derived alleles at SNP sites, V-17.5, V-5, V5.8, V17.8, V25 (10) (Table S1).We then crossed TG3319 males to XSW933 young adult hermaphrodites, and crossed N2 males to CB4856 young adult hermaphrodites as a control.Following successful mating (~12 hours), L4 hermaphrodite worms of the F1 generation from both crosses were individually picked and allowed to grow to the young adult stage.These hermaphrodites were mated to CB5584 males (carrying a pharyngeal GFP marker).After ~12 hours of mating, the F1 animals were plated to lay eggs for two days.F1 animals were picked for single-worm lysis and genotyping by PCR.From the F1 plates that were confirmed to be heterozygous for the CB4856-derived alleles of the chromosome V SNP markers , 256 F2 cross-progeny L4 hermaphrodites (expressing the pharyngeal GFP marker) were picked.Single worm lysis was carried out and the V-17.5, V-5, V5.8, V17.8, V25 SNP sites were tested for presence of the CB4856-derived alleles by PCR.The primers used for the PCR are indicated in Table S1.The PCR reaction was set up at 94°C for 40 seconds, followed by 35 cycles of 40 seconds at 60°C and 1 minute at 72°C.This was followed by 10 minutes at 72°C.PCR products were digested with Takara Quickcut TM Dra I (Aha III) restriction enzyme according to the manufacturer's instruction, then resolved on a 2% agarose gel (Sangon Biotech) for 40 minutes.

Supplemental References
Table S1: Dra I SNP locations and primers sequences for CO distribution assay.

Fig S4 FigFigFigFig
Fig S4 Fig S9 % of oocytes with indicated number of DAPI bodies

Fig S13 .
Fig S13 Fig S14 Chromsome V domains No CO High CO/kb Low CO/kb High CO/kb No CO

Analysis of ATPase-dead rad-54.B(K244A) mutant
(11) Quantification of diakinesis oocyte karyotypes in indicated genotypes.Number of oocytes assessed: WT, n=125; rad-54.B(gt3379[K244A]), n=126.Statistical significance assessed by Mann-Whitney test; WT vs rad-54.B(K244A), ns (p>0.05).B) Representative max-projected images of WT, rad-54.B(gk340656)(null), and rad-54.B(K244A) whole-mount gonads immunostained for RAD-51.Scale bar represents 20 µm.C) Quantification of RAD-51 foci for genotypes depicted in(B).Each data point represents an individual nucleus, with relative position calculated as the percentage of the length between the distal tip (0%) and the end of pachytene zone (100%); different colors represent the different gonads used for quantification.D) Max-projected images of germ cell nuclei from the region of the gonad spanning entry into meiotic prophase.SUN-1 pS8 signal at the nuclear envelope (primarily seen in nuclei on the right side of each image) reflects activation of CHK-2 and marks entry into meiotic prophase, whereas nuclei lacking SUN-1 pS8 signal have not yet entered meiotic prophase.Although meiotic RAD-51 foci were not elevated in the rad-54.B(K244A) mutant (see also E, F below), B-D illustrate variable detection of elevated premeiotic RAD-51 foci in this mutant.Observation of elevated (premeiotic) RAD-51 foci in the rad-54.B(K244A) mutant relative to the rad-54.B(null) mutant is reminiscent of a previously-published report showing that an ATPase-dead allele of yeast rdh54(K352R) causes a stronger DSBR defect than an rdh54(null) mutation(11); both observations suggest that the presence of a catalytically-inactive version of a RAD54B ortholog can in some contexts represent a greater impediment to DSBR than when the protein is absent.Scale bar represents 5 µm.E) Max-projected images of early pachytene nuclei stained for RAD-51 for indicated genotypes.Scale bar represents 5 µm.F) Quantification of early pachytene RAD-51 foci (12)S14.The rad-54.B(null) mutant exhibits a modestly altered distribution of COs.Top: schematic of chromosome V, with chromosome domains as defined by recombination assays(12)depicted by the colored boxes, and SNP markers used indicated with arrowheads below.Bottom: numbers of COs detected in each interval, and numbers of non-CO (NCO) products, from two independent experiments.P-value comparing CO distribution in WT vs rad-54.B(gk340656)(null) was calculated using a chi-square test for independence.