Neale, M. J., Pan, J. & Keeney, S. Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053-1057

Molecular Biology Programs, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Nature (Impact Factor: 41.46). 09/2005; 436(7053):1053-7. DOI: 10.1038/nature03872
Source: PubMed


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.


Available from: Matthew J Neale, Feb 02, 2015
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    • "Immunoprecipitation (IP) was performed using previously described techniques (Neale et al. 2005; Lange et al. 2011). In brief, testes from 45-dpp Spo11 +/− , Tg(Spo11) +/− , and Spo11 −/ − mice were decapsulated and lysed in 800 μl lysis buffer (1 % "
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    ABSTRACT: Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 (+/-) spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 (-/-) background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination.
    Chromosoma 10/2015; DOI:10.1007/s00412-015-0544-7 · 4.60 Impact Factor
    • "Spo11 is orthologous to the topoVI family of topoisomerase discovered in archaea and consistently introduces DSBs by coupled transesterification reactions to form covalent tyrosyl- DNA linkages at the 5 0 termini of the broken DNA. Spo11 is then removed by endonucleolytic cleavage (Neale et al. 2005), liberating short Spo11-DNA oligonucleotide complexes and resected strands, which are further extended to generate recombinogenic 3 0 single-stranded tails. Over the years, meiotic DSBs have been mapped and quantified in yeast genomic DNA using a variety of approaches, including Southern blot analysis of chromosomal fragments or Figure 1. "
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    ABSTRACT: Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalyzed by the evolutionary conserved Spo11 protein and accessory factors. DSBs are nonrandomly distributed along the chromosomes displaying a significant (~400-fold) variation of frequencies, which ultimately establishes local and long-range “hot” and “cold” domains for recombination initiation. This remarkable patterning is set up within the chromatin context, involving multiple layers of biochemical activity. Predisposed chromatin accessibility, but also a range of transcription factors, chromatin remodelers, and histone modifiers likely promote local recruitment of DSB proteins, as well as mobilization, sliding, and eviction of nucleosomes before and after the occurrence of meiotic DSBs. Here, we assess our understanding of meiotic DSB formation and methods to change its patterning. We also synthesize current heterogeneous knowledge on how histone modifications and chromatin remodeling may impact this decisive step in meiotic recombination. © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
    Cold Spring Harbor perspectives in biology 05/2015; 7(5):a016527. DOI:10.1101/cshperspect.a016527 · 8.68 Impact Factor
    • "These include polynucleotide kinase 3' phosphatase (PNKP) [Jilani et al., 1999], Ku itself which can excise nucleotide damage in the vicinity of DNA ends [Roberts et al., 2010], the neurogenerative disease protein aprataxin which removes adenylate groups [Ahel et al., 2006] and the phoshodiesterases TDP1 and 2 which remove DNA topoisimerase adducts [Cortes Ledesma et al., 2009]. In addition, damaged DNA ends can be trimmed by endonucleases such as Artemis or Metnase [Mohapatra et al., 2013], the Aprataxin and PNK-like factor (APLF) [Kanno et al., 2007], the RecQ helicases WRN and BLM in cooperation with the helicase/endonuclease DNA2 [Sturzenegger et al., 2014] and the MRE11/ RAD50/NBS1 (MRN)/CtIP complex [Neale et al., 2005], to make break ends ligatable. Especially the MRN/CtIP complex has been implicated in DSB sensing, initiating the resection of the 5'-DNA strand to produce 3'-single- strand DNA overhangs and facilitate the search for sequence homology required for homology-dependent repair. "
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    ABSTRACT: The discovery of DNA damage response proteins such as γH2AX, ATM, 53BP1, RAD51, and the MRE11/RAD50/NBS1 complex, that accumulate and/or are modified in the vicinity of a chromosomal DNA double-strand break to form microscopically visible, subnuclear foci, has revolutionized the detection of these lesions and has enabled studies of the cellular machinery that contributes to their repair. Double-strand breaks are induced directly by a number of physical and chemical agents, including ionizing radiation and radiomimetic drugs, but can also arise as secondary lesions during replication and DNA repair following exposure to a wide range of genotoxins. Here we aim to review the biological meaning and significance of DNA damage foci, looking specifically at a range of different settings in which such markers of DNA damage and repair are being studied and interpreted. Environ. Mol. Mutagen., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 03/2015; 56(6). DOI:10.1002/em.21944 · 2.63 Impact Factor
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