HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.
To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.
Carty
M P
MP
Section on Viruses and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Levine
A S
AS
Dixon
K
K
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis*; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA; D004257:DNA Polymerase II