Completion of mammalian lagging strand DNA replication using purified proteins.
We have modeled the reactions involved in the completion of lagging strand DNA replication using a synthetic DNA substrate and purified enzymes from calf thymus. We have demonstrated that each polymerase, alpha, delta, and epsilon, is capable of extending an upstream 3' terminus to generate a nick in the DNA substrate that is subsequently ligated by DNA ligase I. Synthesis by each polymerase and subsequent ligation occurred efficiently after the addition of the 50-kDa 5'- to 3'-exonuclease. Analyses are presented which show that a substantial proportion of the ligated products is the result of polymerase epsilon, exonuclease, and DNA ligase I, all acting on the same DNA template. That is, polymerase fills in the gap, then both the polymerase and exonuclease act, one adding and the other removing nucleotides, followed by ligation. Results presented suggest that polymerase alpha, delta, or epsilon may functionally interact with DNA ligase I and the 5'- to 3'-exonuclease to perform the enzymatic reactions required for the completion of lagging strand DNA synthesis.
Turchi
J J
JJ
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Bambara
R A
RA
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D011088:DNA Ligases; D000072481:DNA Ligase ATP