Processivity of the herpes simplex virus DNA polymerase: Role of high-affinity DNA binding by the processivity subunit.

摘要

The herpes simplex virus (HSV) DNA polymerase is a heterodimer of the products of the viral UL30 and UL42 genes. The UL30 gene product, also known as Pol, possesses the catalytic nucleotidyltransferase activity of the polymerase. Without UL42, however, Pol will incorporate only a few nucleotides before dissociating from DNA. UL42 is the processivity subunit of the polymerase; when bound to UL42, Pol incorporates thousands of nucleotides without dissociating. UL42 binds DNA with a dissociation constant in the nanomolar range, and has been proposed to act as a monomer. Thus, UL42 differs from all known non-herpesviral processivity factors, and must confer processive DNA synthesis on its cognate polymerase via a novel mechanism.; This thesis addresses several questions regarding DNA binding by UL42. I first used chemical crosslinking and mobility-shift assays to confirm that UL42 is a monomer when bound to DNA. I then used nuclease footprinting and mobility-shift assays to demonstrate that the placement of protein barriers at the ends of a linear DNA molecule inhibits dissociation of UL42 from the DNA. Moreover, on short DNA molecules, the half-life of UL42 is dependent on the length of the DNA. Both of these observations indicate that UL42 can diffuse to the end of the DNA before dissociating. A calculation of the diffusion coefficient suggests that it is sufficiently rapid to maintain the translocation of UL42 without inhibiting Pol movement, thereby providing an explanation for how UL42 binds DNA with high affinity without slowing the translocation of Pol. Finally, I show that reducing the net positive charge on a proposed DNA interface of UL42 via site-directed mutagenesis reduces DNA binding without affecting Pol binding. These mutations also reduce long-chain DNA synthesis by the Pol:UL42 holoenzyme. Taken together, these results support a model in which a strong electrostatic interaction between UL42 and DNA is sufficient to increase the processivity of the polymerase without slowing the rate at which it synthesizes DNA.