Analysis of DNA Replication Functions of DNA Damage-Kinase Site Mutants of SV40 Large T Antigen

Simian Virus 40 (SV40), a polyomavirus (PyV) with a small, double-stranded DNA (dsDNA) genome, is a historically important model system for several eukaryotic cellular processes. SV40 is closely related to human PyVs JCPyV and BKPyV which have significant impact on human health. SV40 DNA replication in the human host requires only a single viral enzyme, Large T antigen (LT), which acts as the origin binding protein and the replicative DNA helicase. All remaining replication factors are recruited from the host cell. SV40 DNA replication is arrested following several types of DNA damage that also arrest host cell DNA replication, making it an excellent model to study the cellular DNA Damage Response (DDR) pathways which play a pivotal role in the regulation and development of human cancers. Following dsDNA breaks, replication of SV40 genomes is arrested and SV40 LTag is phosphorylated in a manner dependent on one of the major transducer kinases of the human DDR, either ATM or ATR. Thus, the arrest of SV40 DNA replication following DDR is likely due to phosphorylation of LTag. Amino acid alignments of seven mammalian polyomavirus LTags identified a highly conserved and uncharacterized putative DDR kinase recognition site (serine/threonine-glutamine (S/TQ)). Phospho-mimetic (aspartic acid) and non-phosphorylatable (alanine) mutations were created at this threonine residue in LTag and evaluated using in vivo SV40 DNA replication assays. The phospho-mimetic aspartic acid LTag mutant resulted in dramatic inhibition of SV40 DNA replication, while the alanine mutant supports WT levels of DNA replication in vivo. We have expressed and purified WT and T518D mutant LT and shown that the phospho-mimetic mutant is deficient for SV40 DNA replication in vitro. Having purified enzymes in hand allowed us to use an extensive collection of in vitro assays to biochemically dissect the SV40 DNA replication-related functions of LTag. Our analyses indicate that T518D is a defective dsDNA helicase that is unable to efficiently unwind increasing longer dsDNA substrates. Aspartic acid mutations to corresponding residues in JCPyV and BKPyV LT validate our SV40 data and this highly conserved site as an important for controlling PyV DNA replication. Though multiple attempts at identifying DDR-dependent phosphorylation sites on LT were unsuccessful, continued mass spectrometry approaches will confirm T518 phosphorylation and identify factors recruited for and phosphorylated during SV40 DNA replication as a means to contribute to our understanding of higher eukaryotic DNA replication fork dynamics following DNA damage.