The three-dimensional structure and functional studies of the human cytomegalovirus DNA polymerase subunit, UL44

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44. UL44 represents the putative processivity factor for cytomegalovirus since it shares certain features with the herpes simplex virus processivity factor, UL42. However, the properties of UL44 remain poorly characterized in comparison to UL42. This thesis addresses the three-dimensional structure of UL44 and describes biochemical and molecular genetic studies aimed at understanding the oligomeric state of UL44, its DNA binding activity, and its physical association with UL54.; The crystal structure of residues 1--290 of UL44 was solved to 1.85 A resolution. This region of UL44 displays all known biochemical properties of UL44 in vitro. UL44 forms a head-to-head C-clamp shaped homodimer with a central cavity of sufficient size to accommodate double-stranded DNA. Each monomer of UL44 shares a remarkably similar fold to UL42, which is a monomer, and PCNA, which is a trimer, even though these proteins have no obvious sequence homology. Analytical ultracentrifugation and gel filtration studies showed that UL44 also dimerizes in solution. The dimerization activity of UL44 correlates with its ability to bind DNA inasmuch as mutations that reduced dimerization also decreased DNA binding. UL44 appears to act as a hybrid processivity factor as it can partially surround DNA, like PCNA (topological tethering), and bind directly to DNA (electrostatic tethering).; A second structure of UL44 was obtained in complex with the C-terminal 22-residues of UL54 to 2.5 A resolution. Consistent with biochemical and molecular genetic studies, the extreme C terminus of UL54 binds directly to the connector loop of UL44 and an adjacent hydrophobic crevice. The UL44-peptide structure displays a more 'open' conformation than the uncomplexed structure; the possible implications of this conformational change are discussed.