The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding

Abstract

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.Most replicative DNA polymerases depend on accessory proteins known as processivity factors to achieve prolonged association with DNA. In this way polymerases are able to synthesize long stretches of DNA before dissociating from their templates. The most studied processivity factors include proliferating nuclear antigen (PCNA) 3 from eukaryotes and archaebacteria (1, 2), the ␤-subunit from Escherichia coli (3), and gp45 from T4 and RB69 bacteriophage (4, 5). These ring shaped proteins, also known as "sliding clamps," cannot bind DNA on their own and require protein complexes called clamp loaders to be loaded onto DNA as toroidal homomultimers in an ATP-dependent manner (6). They are then able to physically tether the catalytic subunit of polymerase, thus ensuring its processivity (7-9).The DNA polymerase encoded by herpes simplex virus (HSV) is a heterodimer composed of a catalytic subunit, UL30, and a processivity subunit, UL42 (10 -12). UL42 interacts with the C terminus of UL30, increases affinity of the polymerase for primer/template DNA, and stimulates long chain DNA synthesis (13-18). In contrast to sliding clamps, UL42 binds DNA directly as a monomer with nanomolar affinity and does not require ATP hydrolysis or accessory proteins for binding (10,14,18,19). Even though UL42 binds DNA tightly, it is able to diffuse on DNA and does not impede UL30 elongation (18,20).The crystal structure of UL42 has been solved in complex with the C-terminal 36 residues of UL30 (21). The structural fold of UL42 is remarkabl...

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