Reconstitution of uracil DNA glycosylase initiated base excision repair in herpes simplex virus‐1

Abstract

Herpes simplex virus‐1 is self‐sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV‐1 DNA polymerase (UL30) exhibits AP and 5′‐deoxyribose phosphate lyase activities that are integral to base excision repair (BER). We have mapped the lyase activity to the C‐terminal polymerase domain of UL30. This domain lacks polymerase activity but binds DNA. Candidate lysine residues in this fragment will be mutagenized to identify the lyase active site. We have also reconstituted a system with purified HSV‐1 and human proteins that performs all the steps of uracil DNA glycosylase initiated BER. In this system, nucleotide incorporation is dependent on the HSV‐1 uracil DNA glycosylase, AP endonuclease and UL30. Completion of BER is mediated by DNA ligase IIIα/XRCC1, which also confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV‐1 DNA polymerase processivity factor and prevents BER from occurring with heterologous DNA polymerases. These findings demonstrate that the HSV‐1 proteins in combination with cellular factors are capable of performing BER and have implications on viral genome maintenance during lytic replication and reactivation from latency.This work was supported by grant GM 062643 from the NIH.