DNA helicase E and DNA polymerase epsilon functionally interact for displacement synthesis.
A functional interaction between DNA helicase E and DNA polymerase epsilon from calf thymus has been detected which results in the extension of an upstream 3' OH through a downstream primer to the end of a synthetic template. DNA synthesis resulting in full-length extension products was dependent on the addition of DNA helicase E and hydrolysis of ATP, suggesting that displacement of the downstream primer was required. Identical reactions using DNA polymerases alpha and delta in place of DNA polymerase epsilon showed no full-length products dependent on helicase E, indicating that polymerases alpha and delta were incapable of functionally interacting with the helicase. The reaction leading to full-length extension products was time dependent and dependent on the concentration of added polymerase epsilon and helicase E. Exonucleolytic degradation of the downstream primer, or ligation of the downstream primer to the upstream 3' OH, were not responsible for the full-length products observed. Displacement of the downstream primer by DNA helicase E was not affected by the addition of polymerase epsilon to the reactions. Template dilution experiments demonstrated that DNA polymerase epsilon and helicase E were acting in concert to perform displacement synthesis. Additional evidence for functional coordination was obtained by demonstration that DNA helicase E stimulated DNA polymerase epsilon in a standard DNA synthetic assay using dA3000.dT16 as the template-primer. The results presented are consistent with the hypothesis that DNA helicase E and DNA polymerase epsilon are capable of coordinated activities that result in displacement synthesis. A functional interaction of this sort may be involved at the eukaryotic replication fork or in DNA repair.
Turchi
J J
JJ
Department of Biochemistry and Cancer Center, University of Rochester School of Medicine and Dentistry, New York 14642.
Siegal
G
G
Bambara
R A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004265:DNA Helicases / Q000378:metabolism*; D004258:DNA Polymerase III; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases