The papillomavirus E1 helicase and E2 protein bind to and stimulate the enzymatic functions of human topoisomerase I and DNA polymerase delta
Clower, Randolph Vincent
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Progression of eukaryotic DNA replication forks along double-stranded helical DNA occurs through the coordinate action of a replicative helicase, processive polymerase and single-stranded DNA binding protein, in addition to a topoisomerase which produces the relaxed topological DNA substrate. The papillomavirus (PV) E1 helicase, PV E2 transactivation protein, human DNA polymerase delta (pol δ) and human topoisomerase I (topo I) are capable of replicating PV DNA in the presence of other host proteins. PV E1 and E2 are known to play direct roles in recruiting cellular DNA replication factors, such as Replication Protein A (RPA) and DNA polymerase α-primase (pol α/prim) to replicate PV genomes. In this study, PV E1 and E2 were each shown to interact with and stimulate topo I and pol δ. Data is also presented which demonstrates that topo I stimulates E1 binding to the PV origin. The interaction between E1 and topo I was mapped to the DNA binding domain (DBD, as 142-308) and C-terminus (aa 589-649) of E1. PV origin, or non-origin DNA decreased the interaction between full length E1 or the E1 DBD and topo I, whereas the C-terminal interaction domain was unaffected for topo I binding in the presence of DNA. Using topo I DNA relaxation assays, E1 stimulated topo 16- to 7-fold, while E2 stimulated topo 13- to 4-fold. The stimulation of topo I by E1 or E2 is independent of a PV origin. When E1 and E2 were added to a topo I DNA relaxation assay together, their stimulatory affects were not additive. Topo I was also shown to slightly decrease the helicase activity of E1. These findings imply a mechanism for the recruitment of topo I to PV DNA replication forks, and a mechanism for stimulating topo I to allow for efficient relaxation of the torsional stress induced by replication fork progression. Using template elongation assays, E1 and E2 were each also capable of stimulating pol δ enzymatic activity. Using short homopolymer templates, E1 increased pol δ processivity much like Proliferating Cell Nuclear Antigen (PCNA), while E2 stimulated DNA loading of pol δ. When using a longer natural template, addition of E1 to the pol δ holoenzyme, which included PCNA and Replication Factor C (RFC), resulted in up to a 36-fold stimulation. Addition of E2 to the pol δ holoenzyme also increased the activity of pol δ. While single-stranded DNA binding proteins, such as RPA or SSB, are known to be important for optimal pol δ holoenzyme function, addition of E1 to the holoenzyme eliminates the need for RPA. Unlike topo I, the coordination between E1 and pol δ is mutual, as pol δ also stimulates E1's DNA helicase activity. These results demonstrate that PV E1 and E2 are directly linked to the DNA replication elongation apparatus. Culminating this work, a model is presented in which the described interactions assemble a PV DNA replication-elongation apparatus.