The integrity of the genetic material is important for accurate transmission of DNA from cell to cell.  DNA mutations can arise from errors during polymerase-mediated DNA replication; for example during translesion DNA synthesis (TLS), which involves specialized polymerases that replicate past replication-blocking DNA lesions.  While many studies investigate nucleoside triphosphate insertion opposite DNA lesions, less is known regarding mutagenicity for the extension process immediately following lesion bypass, i.e. post-lesion DNA synthesis (PLS).  In this study, 3' synthetic base-modified primers that paired opposite the model lesion O6-benzylguanine (O6-BnG) were used as novel molecular probes to investigate PLS catalysed by the error-prone DNA polymerase IV (Dpo4) from Sulfolobus solfataricus.  Single nucleotide incorporation, full length primer extension, steady-state kinetic incorporation efficiencies, and computational modeling of base-modified primers within the active site of Dpo4 will be presented.  Understanding mechanisms of PLS can deepen our understanding of how mutations are acquired and O6-alkylguanine adduct processing.

Mechanism of post-lesion DNA synthesis with an error-prone DNA polymerase probed with 3' synthetic base-modified primers

244th National Meeting (2012)

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