Effects of Substitutions of Arginine Residues on the Basic Surface of Herpes Simplex Virus UL42 Support a Role for DNA Binding in Processive DNA Synthesis

Abstract

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14-to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.Replicative DNA polymerases are highly processive enzymes that synthesize long stretches of DNA without dissociating from the template. Many replicative polymerases depend on accessory proteins called processivity factors, which confer processivity on their cognate polymerases by slowing their dissociation from DNA. Among these are the "sliding clamp" processivity factors (18), exemplified by proliferating cell nuclear antigen (PCNA), the processivity factor for eukaryotic replicative DNA polymerases. All sliding clamps adopt a common structure, in which a dimer or trimer of the sliding clamp protein forms a ring that encircles DNA (20,21,23,28). The association of the sliding clamps with DNA requires the activity of accessory proteins known as clamp loaders (19), which open the clamp ring and reclose it on DNA in an ATP-dependent reaction. The sliding clamp then can tether the catalytic polymerase core to DNA, thus ensuring processivity without impeding the movement of the polymerase.The DNA polymerase that replicates herpes simplex virus (HSV) is a heterodimer of a 137-kDa catalytic subunit (UL30, also called Pol) and a 60-kDa processivity subunit, UL42 (15,24,25). The extreme C terminus of Pol is necessary for interaction with UL42, long-chain DNA synthesis, and viral re...

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

“…6, lanes 3, 7, 9, and 11, respectively). It should be noted that all of the mutants with decreased binding affinity for DNA are not defective in the ability to increase the polymerase processivity, in contrast to the observations for UL42 (39,40) and UL44 (41). In particular, the C95E mutant, which is defective in dimer formation as well as in DNA binding activity, efficiently increased the polymerase processivity, strongly suggesting that the monomeric form of the BMRF1 protein interacts with the BALF5 polymerase catalytic subunit to function as a polymerase processivity factor.…”

Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

“…Moreover, UL42 appears to facilitate the loading of the polymerase onto DNA, to increase the preference of polymerase for binding to primer/template junctions, and to slow the dissociation of polymerase from DNA without reducing its elongation rate (38,39). Amino acid substitutions on the basic surface of the UL42 protein, which specifically disrupt DNA binding, negatively affect processivity (40). Thus, the DNA binding properties of UL42 are central for its role in processivity.…”

Herpes Simplex Virus Mutants with Multiple Substitutions Affecting DNA Binding of UL42 Are Impaired for Viral Replication and DNA Synthesis

“…Insertion mutations of UL42 that result in undetectable DNA binding impair long-chain DNA synthesis and prevent complementation of a UL42 null mutant (1). UL42 mutants containing alanine substitutions for any of four conserved arginine residues (at positions 113, 182, 279, and 280), which reside on the positively charged surface of UL42, decrease DNA binding without affecting binding to the Pol C terminus and decrease the stimulation of long-chain DNA synthesis (12). However, when incorporated into herpes simplex virus, these single substitutions have only modest effects on viral replication and DNA synthesis (5).…”

“…We constructed two plasmids, pHC-R113/182A and pHC-R279/280A, each of which contains two arginine-to-alanine substitutions, as previously described using pHC700 (5) and plasmids based on pMBP-pp⌬340 containing the corresponding mutations (12). Each plasmid was sequenced to confirm the presence of the desired mutations and no others.…”