Crossovers are associated with mutation and biased gene conversion at recombination hotspots

Abstract

Meiosis is a potentially important source of germline mutations, as sites of meiotic recombination experience recurrent double-strand breaks (DSBs). However, evidence for a local mutagenic effect of recombination from population sequence data has been equivocal, likely because mutation is only one of several forces shaping sequence variation. By sequencing large numbers of single crossover molecules obtained from human sperm for two recombination hotspots, we find direct evidence that recombination is mutagenic: Crossovers carry more de novo mutations than nonrecombinant DNA molecules analyzed for the same donors and hotspots. The observed mutations were primarily CG to TA transitions, with a higher frequency of transitions at CpG than non-CpGs sites. This enrichment of mutations at CpG sites at hotspots could predominate in methylated regions involving frequent single-stranded DNA processing as part of DSB repair. In addition, our data set provides evidence that GC alleles are preferentially transmitted during crossing over, opposing mutation, and shows that GC-biased gene conversion (gBGC) predominates over mutation in the sequence evolution of hotspots. These findings are consistent with the idea that gBGC could be an adaptation to counteract the mutational load of recombination.

Keywords: biased gene conversion; crossover; meiotic recombination; mutation; sequence evolution.

Fig. 1.

COs, mutations, and CCOs in HSI. (A) Distribution of both reciprocal COs in HSI (marks represent different donors). A best-fit normal distribution (Gaussian function) of the CO breakpoints represents the hotspot center at x_c (vertical line), verified by data representing the DSB region (33), shaded in gray, and the Myers motif (allowing one mismatch or less) (1) represented as crosses on the x-axis. (B) Distribution of mutations. The sequenced region (yellow shaded area) shows the new mutations with red crosses (asterisk denotes a CpG) on different haplotypes (mutations per haplotype and donor identification shown on the left). Black and white circles denote heterozygous SNPs (red rim = AT-Weak alleles; black rim = GC-Strong alleles), and gray circles are homozygous SNPs. The vertical dotted line shows the hotspot center. (C) Distribution of CCOs. Different CCOs identified in the same donors as above (frequency of each CCO per haplotype to the left). CCOs are within 60 bp of another heterozygous site in 56% of the cases, suggesting conversion tracks in CCOs involved a single SNP, although the SNP involved cannot be determined unequivocally (SI Appendix, Table S4).

Fig. 2.

Model of CO-driven evolution. (A) Mutagenic model. Mutagenic activity of recombination could be associated to the deamination of methyl-C at a CpG site during 3′-end resection and single-stranded DNA formation, which introduces a thymine that remains unrepaired. In addition to deamination of CpG sites, translesion polymerases may also introduce mutations at hotspots (21) if the repair of heteroduplexes by the mismatch repair machinery active during meiosis is biased towards the newly synthesized strand. (B) gBGC. During the repair of DSBs, mismatches in intermediate heteroduplex tracts at polymorphic sites (triangles) can be either resolved restoring the original allele or can lead to gene conversion (gBGC) favoring GC alleles (red) versus AT alleles (blue). In the case of gBGC, more COs will have breakpoints with GC alleles distal to the DSB than proximal, distorting the segregation ratio of alleles between reciprocals.

Fig. 3.

Transmission distortion between reciprocal COs. (Top) Frequencies of CO breakpoints are compared between reciprocals (blue and orange) in HSI (donors 1042, 1290, 1087, 1050, and 7023) and HSII (donor 1081), A–F, respectively, with numbers representing CO breakpoint counts (nRI vs. nRII) and the position of phased alleles of heterozygous SNPs of NRs shown on top. (Middle) Proportion of CG (S) alleles per heterozygous sites of the donor. (Lower) Log of the rate ratios of the different recombinant haplotypes, calculated as log[(nRI/totalRI)/(nRII/totalRII)], where the denominator is the total number of either COs (black) or meiosis (red) surveyed per reciprocal. Asterisks denote significant transmission distortion, based on the standardized Pearson residual (black asterisks denote the haplotype with the strongest evidence of heterogeneity; SI Appendix, Table S3). Note that for HSII, the largest skew occurred at an indel polymorphism of a homopolymeric run of six or seven consecutive As in donor 1081.