PCNA-induced DNA synthesis past cis-syn and trans-syn-l thymine dimers by calf thymus DNA polymerase δ in vitro

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-l cyclobutane thymine dimers by calf thymus DNA polymerase δ (Polδ) in vitro. Templates containing site-specific cis-syn and trans-syn-l thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA lligation. Extension of a 15-mer primer on the UV dlmer-containing templates by Polδ 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 _μM, Polδ could not bypass the cis-syn dlmer and terminated elongation one nucleotkle prior to the 3-T of the dimer. DNA synthesis past the trans-syn-l dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotlde prior to the 3′-T of both dimers at 1 _μM dNTPs but opposite the 5′-T of the dimers at 100 _μM 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-l dlmer.