Methods to Study DNA End Resection I: Recombinant Protein Purification
METADATA ONLY
Loading...
Author / Producer
Date
2018
Publication Type
Book Chapter
ETH Bibliography
yes
Citations
Altmetric
METADATA ONLY
Data
Rights / License
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.
Permanent link
Publication status
published
Book title
Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination
Journal / series
Volume
600
Pages / Article No.
25 - 66
Publisher
Academic Press
Event
Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
Protein purification; Homologous recombination; DNA end resection; Nuclease; Helicase; Biochemistry; Mre11; Dna2; Bloom; Werner