Interaction of proliferating cell nuclear antigen with yeast DNA polymerase delta.
Previously, we described the purification of the catalytic core subunit of yeast DNA polymerase delta from Escherichia coli carrying the yeast POL3 gene cloned in a vector that allowed efficient expression. The basic catalytic properties of the single subunit enzyme are virtually identical to the heterodimeric core enzyme purified from yeast cells (Brown, W. C., Duncan, J. A., and Campbell, J. L. (1993) J. Biol. Chem. 268, 982-990). In this work, we investigate the ability of yeast proliferating cell nuclear antigen (PCNA) to stimulate processive synthesis by the bacterially produced, single-subunit DNA polymerase delta. Yeast PCNA was found to stimulate the full-length single-subunit yeast DNA polymerase delta and to increase its processivity. A truncated version of DNA polymerase delta, from which the NH2-terminal 220 amino acids had been removed, was prepared and similarly investigated. While the catalytic properties of the truncated protein were nearly identical to those of the full-length enzyme, neither the extent of synthesis nor processivity was increased in the presence of PCNA. Thus, we conclude that the single-subunit DNA polymerase can associate productively with PCNA in the absence of other proteins and that the NH2-terminal domain of the catalytic subunit must be intact for this interaction.
Brown
W C
WC
Braun Laboratories, California Institute of Technology, Pasadena 91125.
Campbell
J L
JL
D000818:Animals; D002417:Cattle; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; C016069:poly (dA).oligo((d)T); D011067:Poly dA-dT; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase