Coupling of DNA helicase function to DNA strand exchange activity.

Content Provider	Semantic Scholar
Author	Harmon, F. G. Kowalczykowski, Stephen C.
Copyright Year	2000
Abstract	1. Introduction DNA repair can occur by a variety of mechanistically distinct pathways [for review, see (1)]. Recombinational DNA repair is one such pathway, and it requires the coordinated action of many different enzymes. In the best studied organism, Escherichia coli, more than 20 different proteins are involved [for review, see (2)]. The recombinational repair of a double-stranded DNA (dsDNA) break requires four general steps: (1) processing; (2) homologous pairing; (3) DNA heteroduplex extension; and (4) resolution. Here we describe assays to study aspects of the first two steps. One of the most important proteins for DNA repair by homologous recom-bination is the RecA protein [for review, see (3–5)]. RecA-like proteins are highly conserved and are present in all organisms thus far examined (6). Loss of RecA protein function renders E. coli cells highly sensitive to DNA damaging agents (7), and reduces recombination frequency to 0.1% of wild-type levels (8). In vitro, RecA protein promotes both the pairing and exchange of DNA strands between two homologous DNA molecules, provided that one of them contains a region of single-stranded DNA (ssDNA). RecA protein assembles on this ssDNA to form a nucleoprotein filament, referred to as the presynaptic complex. It is this nucleoprotein filament that is active for homologous pairing and DNA strand exchange (9). In vivo, most DNA breaks do not directly produce a ssDNA substrate for RecA protein, thus necessitating processing of the lesion by other enzymes (1). In most cases, damage-specific DNA helicases process the DNA break to produce a region of ssDNA sufficient for binding by RecA protein (2). In E. coli, dsDNA breaks are acted upon by RecBCD enzyme, a DNA helicase/nuclease
Starting Page	75
Ending Page	89
Page Count	15
File Format	PDF HTM / HTML
Alternate Webpage(s)	http://microbiology.ucdavis.edu/kowalczykowski/PDF_files/Harmon%20and%20Kowalczykowski%20(2000)%20Methods%20in%20Mol.%20Biol.%20152,%2075-89.pdf
PubMed reference number	10957970v1
Volume Number	152
Journal	Methods in molecular biology
Language	English
Access Restriction	Open
Subject Keyword	Caspase-1 Cytoskeletal Filaments DNA Breaks DNA Breaks, Double-Stranded DNA Breaks, Single-Stranded DNA Helicase A DNA Helicases DNA Repair DNA, Single-Stranded Greater Than Nucleoproteins nuclease recombinational repair
Content Type	Text
Resource Type	Article