Biochemical analysis of translesion DNA synthesis in human post-replication repair pathway

The purpose of author's experiments described here is to elucidate the molecular mechanism of DNA polymerase change to regulate the occurrence of mutation minimal. The change occurs at translesion DNA synthesis (TLS) in the post-replication repair pathway mutually between the error-free DNA polymerase (polδ) and error-prone Y-pol: the latter Y-pol can repair at the damaged base, which frequently results in mutation. In a series of experiments below, authors obtained needed protein components mainly from their over-expressing E. coli. First, the system to synthesize DNA with polδ was constructed, which consisting from the enzyme, single strand DNA as a template, replication protein A (RPA), PCNA (proliferating cell nuclear antigen), replication factor C (RFC) and substrate nucleotides. The efficient DNA synthesis was found to occur and properties of the protein components were characterized. Second, the reaction system of PCNA ubiquitination was established in vitro, which consisting from the ubiquitin (UBQ) activating enzyme E1, RAD6-RAD18 complex, UBQ, template DNA coated by RFC and RPA, to characterize their properties; and the reaction was found for recognition factor of DNA damage to be unnecessary as it proceeded regardless to DNA synthesis. Third, using the template DNA damaged by UV, polδ and Polη (a pol-Y) as a TLS polymerase, examined was if the dynamic, ubiquitinated PCNA promoted the polymerase change, polδ-Polη-polδ: elongation of the chain and its stop at the damaged site by polδ, elongation by Polη and then again by polδ, were confirmed. The model sequence suggested that at the stop of elongation, the 3'-OH terminal and Polη assembled to elongate further and afterward, polδ was again selectively in charge of the reaction. The mutual changing of polymerases on DNA damaged by radiation was to be further examined in details. (K.T.)