Completion of mammalian lagging strand DNA replication using purified proteins

Abstract

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.