DNA damaged-induced phosphorylation of a viral replicative DNA helicase results in inhibition of DNA replication through attenuation of helicase function

A major function of the DNA damage responses (DDRs) that act during the replicative phase of the cell cycle is to inhibit initiation and elongation of DNA replication. The polyomavirus SV40 is an important model system for studying human DNA replication and DDRs due to its heavy reliance on host factors for viral DNA replication, and the arrest of SV40 DNA replication in response to DDR activation. The inhibition of SV40 DNA replication following DDR activation is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral origin-binding protein and DNA helicase. NetPhos prediction of LT phosphorylation on multiple sites were confirmed by mass spectroscopy, including a highly conserved DDR kinase site, T518. In cell-based DNA replication assays expression of the phosphomimetic mutant form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication; while LT-dependent transcriptional activation was unaffected. WT and LT T518D were subsequently expressed, purified, and analyzed in vitro for assessment of biochemical function. In concordance with the cell-based data, reactions using SV40 LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication. Importantly, the LT T518D mutation did not affect critical LT protein interactions or its ATPase function, but showed decreased helicase activity on long, but not very short, DNA templates. These results suggest that DDR phosphorylation at T518 inhibits SV40 DNA replication by impeding LT helicase activity, thereby slowing the DNA replication fork. This is consistent with the slowing of cellular replication forks following DDR and may provide a paradigm for another mechanism for how DNA replication forks can be slowed in response to DDR, by phosphorylation of DNA helicases.