PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro.
Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.
Department of Chemistry, Washington University, St Louis, MO 63130.
C L
CL
O'Day
P M
PM
Burgers
J S
JS
Taylor
D000818:Animals; D000951:Antigens, Neoplasm / Q000494:pharmacology; D001324:Autoantigens / Q000494:pharmacology; D001483:Base Sequence; D002417:Cattle; D004258:DNA Polymerase III; D004261:DNA Replication / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis*; D018809:Proliferating Cell Nuclear Antigen; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
D000951:Antigens, Neoplasm; D001324:Autoantigens; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D011740:Pyrimidine Dimers; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase