Resolution of single and double Holliday junction recombination intermediates by GEN1

Punatar, Rajvee Shah; Martín, María J.; Wyatt, Haley D.M.; Chan, Ying Wai; West, Stephen C.

doi:10.1073/pnas.1619790114

Cited by 35 publications

(36 citation statements)

References 68 publications

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“…Rather, our data suggest that the nuclease ensemble processes joint molecule intermediates by biased resolution-independent nicking of dsDNA in the vicinity of HJs. Since HJs are symmetric and their resolution can yield both crossovers and non-crossovers 41 , how is the crossover bias established? As hMSH4-hMSH5 likely stabilizes asymmetric HJ precursors 5 , it is anticipated that the heterodimer will be ultimately present asymmetrically later at the mature joint molecules containing HJs (Extended Data Fig.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the MLH1-MLH3 endonuclease in meiosis

Cannavó¹,

Sanchez²,

Anand³

et al. 2020

Preprint

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32During prophase of the first meiotic division, cells deliberately break their DNA. 33These DNA breaks are repaired by homologous recombination, which facilitates 34proper chromosome segregation and enables reciprocal exchange of DNA seg-35 ments between homologous chromosomes, thus promoting genetic diversity in 36 the progeny 1 . A successful completion of meiotic recombination requires nucleo-37 lytic processing of recombination intermediates. Genetic and cellular data impli-38 cated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in gen-39 erating crossovers, but mechanisms that lead to its activation were unclear 2-4 . 40Here, we have biochemically reconstituted key elements of this pro-crossover 41 pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to 42 support crossing over [5][6][7] , binds branched recombination intermediates and phys-43 ically associates with MutLγ. This helps stabilize the ensemble at joint molecule 44 structures and adjacent dsDNA. Second, we show that MutSγ directly stimulates 45 DNA cleavage by the MutLγ endonuclease, which demonstrates a novel and unex-46 pected function for MutSγ in triggering crossing-over. Third, we find that MutLγ 47 tion of yeast MutLγ is dependent on the integrity of the metal binding 75 DQHA(X)2E(X)4E motif within Mlh3, implicating the nuclease of Mlh3 in resolving 76 recombination intermediates 2,3,18,19,56 . Despite wealth of genetic and cellular data, 77 the mechanisms that control the MutLγ nuclease and lead to biased joint molecule 78 processing remained undefined. 79 80 81 82 83 84 4 Results 85 86 Human MutLγ is an ATP-stimulated endonuclease 87To study human MutLγ (hMLH1-hMLH3), we expressed and purified the hetero-88 dimer from insect cells ( Fig. 1a and Extended Data Fig. 1a,b). Similarly to the mis-89 match repair (MMR)-specific hMutLα (hMLH1-hPMS2) 20 , the hMLH1-hMLH3 90 complex non-specifically nicked double-stranded supercoiled DNA (scDNA) in the 91 presence of manganese without any other protein co-factor ( Fig. 1b,c, Extended 92 Data Fig. 1c), while almost no activity was observed with magnesium (Extended 93 Data Fig. 1d), which is believed to be the specific metal co-factor 20 . Mutations in 94 the conserved metal binding motif of hMLH3 abolished the endonuclease, indicat-95 ing that the DNA cleavage activity was intrinsic to the hMutLγ heterodimer ( Fig. 96 1d, see also Extended Data Fig. 1e). ATP promoted the nuclease activity >2-fold 97 ( Fig. 1d,e, Extended Data Fig. 1f-h). Experiments with various ATP analogs re-98 vealed that ATP hydrolysis by hMLH1-hMLH3 was required for the maximal stim-99 ulation of DNA cleavage (Fig. 1f, Extended Data Fig. 1h). The N-termini of both 100 hMLH1 and hMLH3 proteins contain conserved Walker motifs implicated in ATP 101 binding and hydrolysis 21 . To define whether the ATPase of hMLH1, hMLH3 or 102 both subunits of the heterodimer promotes its nucleolytic activity, we prepared 103 the respective hMutLγ variants with mutations in the conserved motifs of either 104 subu...

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Section: Discussionmentioning

confidence: 99%

Regulation of the MLH1-MLH3 endonuclease in meiosis

Cannavó¹,

Sanchez²,

Anand³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Paralleling these genetic observations, physical examination of HR intermediates during wild‐type yeast meiosis revealed the unexpected existence of multi‐chromatid joint molecules (mcJMs) . Finally, the individual nucleases involved in CO formation are efficient on flap and branched JM substrates, but their activity on HJ/dHJ substrates is weak, with the exception of Yen1/GEN1 and the mammalian SLX1‐4/MUS81‐EME1/XPF‐ERCC1 complex . These additional genetic, physical, and biochemical insights suggest a greater complexity in the processing of DNA strand exchange intermediates formed during meiosis (and mitosis) than previously anticipated, and led to the proposal that at least a subset of COs could arise through dHJ‐independent pathways …”

Section: Implications Of Mir For Allelic and Non‐allelic Homologous Rmentioning

confidence: 91%

Multi‐Invasion‐Induced Rearrangements as a Pathway for Physiological and Pathological Recombination

Heyer

2018

BioEssays

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Cells mitigate the detrimental consequences of DNA damage on genome stability by attempting high fidelity repair. Homologous recombination templates DNA double-strand break (DSB) repair on an identical or near identical donor sequence in a process that can in principle access the entire genome. Other physiological processes, such as homolog recognition and pairing during meiosis, also harness the HR machinery using programmed DSBs to physically link homologs and generate crossovers. A consequence of the homology search process by a long nucleoprotein filament is the formation of multi-invasions (MI), a joint molecule in which the damaged ssDNA has invaded more than one donor molecule. Processing of MI joint molecules can compromise the integrity of both donor sites and lead to their rearrangement. Here, two mechanisms for the generation of rearrangements as a pathological consequence of MI processing are detailed and the potential relevance for non-allelic homologous recombination discussed. Finally, it is proposed that MI-induced crossover formation may be a feature of physiological recombination.

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“…These may form during DSB repair where only one strand invades template DNA, or when one of the D-loop arms has been cleaved prior to second end capture (Wechsler et al 2011;Shah Punatar et al 2017). To date, three structure-specific nucleases capable of processing HJs or similar structures have been identified in eukaryotic cells, including human MUS81-EME1 and yeast Mus81-Mms4, human SLX1-SLX4 and yeast Slx1-Slx4 as well as human GEN1 and yeast Yen1 (Dehe and Gaillard 2017).…”

Section: Resolution Of Double Holliday Junctionsmentioning

confidence: 99%

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

2018

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DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.

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Resolution of single and double Holliday junction recombination intermediates by GEN1

Cited by 35 publications

References 68 publications

Regulation of the MLH1-MLH3 endonuclease in meiosis

Regulation of the MLH1-MLH3 endonuclease in meiosis

Multi‐Invasion‐Induced Rearrangements as a Pathway for Physiological and Pathological Recombination

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

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