Analysis of translesion replication across an abasic site by DNA polymerase IV of Escherichia coli

Unrepaired replication-blocking DNA lesions are bypassed by specialized DNA polymerases, members of the Y superfamily. In Escherichia coli the major lesion bypass DNA polymerase is pot V, whereas the function of its homologue, pot IV, is not fully understood. In vivo analysis showed that pot V has a major role in bypass across an abasic site analog, with little or no involvement of pot IV. This can result from the inability of pot IV to bypass the abasic site, or from in vivo regulation of its activity. In vitro analysis revealed that purified pot IV, in the presence of the P subunit DNA sliding clamp, and the gamma complex clamp loader, bypassed a synthetic abasic site with very high efficiency, reaching 73% in 2 min. Bypass was observed also in the absence of the processivity proteins, albeit at a 10- to 20-fold lower rate. DNA sequence analysis revealed that pot IV skips over the abasic site, producing primarily small deletions. The RecA protein inhibited bypass by pot IV, but this inhibition was alleviated by single-strand binding protein (SSB). The fact that the in vitro bypass ability of pot IV is not manifested under in vivo conditions suggests the presence of a regulatory factor, which might be involved in controlling the access of the bypass polymerases to the damaged site in DNA. (C) 2003 Elsevier B.V. All rights reserved.