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      Concerted cutting by Spo11 illuminates meiotic DNA break mechanics

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          Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.

          Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.
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            Endonucleolytic processing of covalent protein-linked DNA double-strand breaks.

            DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.
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              Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks.

              To repair double-strand DNA breaks by homologous recombination, the 5'-terminated DNA strand must first be resected, which generates 3' single-stranded DNA overhangs. Genetic evidence suggests that this process is initiated by the Mre11-Rad50-Xrs2 (MRX) complex. However, its involvement was puzzling, as the complex possesses exonuclease activity with the opposite (3' to 5') polarity from that required for homologous recombination. Consequently, a bidirectional model has been proposed whereby dsDNA is first incised endonucleolytically and MRX then proceeds back to the dsDNA end using its 3' to 5' exonuclease. The endonuclease creates entry sites for Sgs1-Dna2 and/or Exo1, which then carry out long-range resection in the 5' to 3' direction. However, the identity of the endonuclease remained unclear. Using purified Saccharomyces cerevisiae proteins, we show that Sae2 promotes dsDNA-specific endonuclease activity by the Mre11 subunit within the MRX complex. The endonuclease preferentially cleaves the 5'-terminated dsDNA strand, which explains the polarity paradox. The dsDNA end clipping is strongly stimulated by protein blocks at the DNA end, and requires the ATPase activity of Rad50 and physical interactions between MRX and Sae2. Our results suggest that MRX initiates dsDNA break processing by dsDNA endonuclease rather than exonuclease activity, and that Sae2 is the key regulator of this process. These findings demonstrate a probable mechanism for the initiation of dsDNA break processing in both vegetative and meiotic cells.
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                Journal
                Nature
                Nature
                Springer Science and Business Media LLC
                0028-0836
                1476-4687
                June 09 2021
                Article
                10.1038/s41586-021-03389-3
                34108687
                ca53a468-0ee5-45a7-82b0-162eac8e97ff
                © 2021

                https://www.springer.com/tdm

                https://www.springer.com/tdm

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