RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro.
To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.
Karthikeyan
G
G
Department of Biological Sciences, Tata Institute of Fundamental Research, Bombay, India.
Lakshmikant
G S
GS
Wagle
M D
MD
Krishnamoorthy
G
G
Rao
B J
BJ
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli; D005092:Exonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000378:metabolism*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004247:DNA; D011938:Rec A Recombinases; D004256:DNA Polymerase I; D005092:Exonucleases