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Methods to Study DNA End Resection I: Recombinant Protein Purification


Anand, Roopesh; Pinto, Cosimo; Cejka, Petr (2018). Methods to Study DNA End Resection I: Recombinant Protein Purification. In: Weber, Gert; Bornscheuer, Uwe T; Wei, Ren. Methods in Enzymology. New York: Elsevier, 25-66.

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.

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.

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Language:English
Date:2018
Deposited On:15 Feb 2021 17:24
Last Modified:16 Feb 2021 21:02
Publisher:Elsevier
Series Name:Methods in Enzymology
Number:206
ISSN:0076-6879
ISBN:978-0-12-182107-4
OA Status:Closed
Publisher DOI:https://doi.org/10.1016/bs.mie.2017.11.008
PubMed ID:29458761

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