Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike ''sliding clamp'' processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were Ϸ10 4 -fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 ''hops'' rather than ''slides,'' i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins.herpes simplex virus ͉ linear diffusion D NA polymerases are central to DNA replication. Most replicative DNA polymerases include accessory subunits that promote replication of long stretches of DNA without dissociating from the template. The best known of these processivity factors are the ''sliding clamps'' (reviewed in ref. 1), which include polymerase subunits of bacteria, eukaryotes, and archaea that form multimeric rings around DNA with the aid of ATP-dependent clamp-loaders. These rings then tether their cognate catalytic subunits to DNA, permitting processive DNA synthesis.A variety of cellular and viral polymerases include processivity subunits that do not use ATP or other proteins for loading onto DNA. Of these, herpes simplex virus (HSV) UL42 is one of the best characterized. This protein's structure resembles that of a monomer of the sliding clamp proliferating cell nuclear antigen (2), yet UL42 binds directly to DNA as a monomer with relatively high affinity (apparent dissociation constant (K d ) in the nanomolar range) (3-5). This direct binding of DNA by UL42 tethers the catalytic subunit of HSV DNA polymerase (Pol) to DNA, thereby enabling processivity (3,(5)(6)(7).An important attribute of processivity factors is their ability to diffuse on DNA. Such diffusion permits teth...

bMethylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicellazoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.

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...

Summary Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the “circle.” The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a “hybrid” of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both.