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Methods to Study DNA End Resection I: Recombinant Protein Purification

Published on Jan 1, 2018in Methods in Enzymology1.862
· DOI :10.1016/bs.mie.2017.11.008
Roopesh Anand6
Estimated H-index: 6
(USI: University of Lugano),
Cosimo Pinto5
Estimated H-index: 5
(UZH: University of Zurich),
Petr Cejka28
Estimated H-index: 28
(ETH Zurich)
Abstract
Abstract Accurate repair of DNA double-strand breaks (DSBs) is carried out by homologous recombination. In order to repair DNA breaks by the recombination pathway, the 5′-terminated DNA strand at DSB sites must be first nucleolytically processed to produce 3′-overhang. The process is termed DNA end resection and involves the interplay of several nuclease complexes. DNA end resection commits DSB repair to the recombination pathway including a process termed single-strand annealing, as resected DNA ends are generally nonligatable by the competing nonhomologous end-joining machinery. Biochemical reconstitution experiments provided invaluable mechanistic insights into the DNA end resection pathways. In this chapter, we describe preparation procedures of key proteins involved in DNA end resection in human cells, including the MRE11–RAD50–NBS1 complex, phosphorylated variant of CtIP, the DNA2 nuclease–helicase with its helicase partners Bloom (BLM) or Werner (WRN), as well as the single-stranded DNA-binding protein replication protein A. The availability of recombinant DNA end resection factors will help to further elucidate resection mechanisms and regulatory processes that may involve novel protein partners and posttranslational modifications.
  • References (107)
  • Citations (5)
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References107
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Meiosis is a specialized double cell division that generates haploid gametes from diploid parent cells. Meiotic recombination between homologous chromosomes ensures the proper segregation of chromosomes to the daughter cells. Mimitou et al. carried out a genome-wide survey of resection of the ends of DNA double strand breaks in yeast. Resection generates single-stranded tails that are vital for meiotic recombination. The Tel1 kinase promoted initiation of resection. Nucleosomes, which normally p...
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The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles th...
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Summary The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11 , abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DS...
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To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target m...
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Summary The human Mre11/Rad50/Nbs1 (hMRN) complex is critical for the sensing, processing, and signaling of DNA double-strand breaks. The nuclease activity of Mre11 is essential for mammalian development and cell viability, although the regulation and substrate specificity of Mre11 have been difficult to define. Here we show that hMRN catalyzes sequential endonucleolytic and exonucleolytic activities on both 5′ and 3′ strands of DNA ends containing protein adducts, and that Nbs1, ATP, and adduct...
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The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components,...
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During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the puta...
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BLM or WRN helicases function with the DNA2 helicase-nuclease to resect DNA double-strand breaks and initiate homologous recombination. Upon DNA unwinding by BLM/WRN, RPA directs the DNA2 nuclease to degrade the 59-strand, revealing the 39 overhang needed for recombination. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single molecule biochemistry, we show that phosphorylated CtIP dramatical...
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Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activit...
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Abstract DNA end resection initiates DNA double‐strand break repair by homologous recombination. MRE11‐RAD50‐NBS1 and phosphorylated CtIP perform the first resection step via MRE11‐catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae , is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, func...
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