Claeys Bouuaert, C. | Search bioRxiv

bioRxiv2017

Rahu Is A Mutant Allele Of Dnmt3c, Encoding A DNA Methyltransferase Required For Meiosis And Transposon Repression In The Mouse Male Germline

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs--Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l--that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called rahu, which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function in vivo. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.

genetics↗

bioRxiv2017

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

Mlh1-Mlh3 (MutL{gamma}) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutL{gamma} has DNA binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutL{gamma}. Limited proteolysis and atomic force microscopy showed that purified recombinant MutL{gamma} undergoes ATP-driven conformational changes. In vitro, MutL{gamma} displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutL{gamma} bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutL{gamma} for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutL{gamma}, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1{Delta}, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3{Delta} caused more chromosome missegregation than mlh1{Delta}, whereas mlh1{Delta} but not mlh3{Delta} partially alleviated meiotic defects of msh5{Delta} mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutL{gamma} complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.\n\nAuthor summarySexual reproduction involves the fusion of two gametes that each contain half of the DNA from each parent. These gametes are generated through a specialized cellular division called meiosis. During meiosis, the cell faces the challenge of identifying the appropriate pairs of chromosomes that need to be separated. This involves an elaborate mechanism whereby the parental chromosomes recombine and form crossovers, i.e. exchange DNA fragments. These crossovers are thus important for the accurate segregation of chromosomes and are also fundamental to evolution because they help shuffle linkage groups from one generation to another. Here, we have studied a complex of proteins called MutL{gamma} that is important for the formation of crossovers, and is also involved in an unrelated mechanism that repairs mistakes that spontaneous arise in DNA when it is synthesized. We uncovered intriguing features of the interaction of this complex with DNA. In addition, by studying a collection of mutants of MutL{gamma}, we identified mutants that affect one biological function but not another. For example, surprisingly, we found mutations that decrease the frequency of crossovers but did not affect chromosome segregation as much as expected. Taken together, our findings allow us to reconsider the ways in which we think about these processes.

genetics↗