The C-Terminal Third of UL42, a HSV-1 DNA Replication Protein, Is Dispensable for Viral Growth

Gao, Min; DiTusa, Sandra Feuer; Cordingley, Michael G.

doi:10.1006/viro.1993.1304

Cited by 28 publications

(24 citation statements)

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“…This region encompasses the minimal Pol-binding domain of UL42 (amino acids 1 to 315) as shown by coimmunoprecipitation studies (Tenney et al, 1993b). This region also encompasses a region of UL42 (amino acids 1 to 348) that has recently been shown to have all the functions required for a productive viral infection in tissue culture (Gao et al, 1993). In this study, we have also used co-immunoprecipitation with Pol to show that the 28K and 8K polypeptides complex with Pol.…”

Section: R K Hamatake and Othersmentioning

confidence: 92%

“…The preponderance of Pol as the Pol/UL42 complex within infected cells suggests that the ability of UL42 to complex with Pol is of critical importance during HSV-1 replication. In support of this hypothesis are recent genetic data that correlate the ability of mutant UL42 proteins to bind Pol with the ability to support viral growth by either complementation of a UL42 null mutant (Digard et al, 1993b) or isolation of UL42 deletion mutants (Gao et al, 1993). Similarly, Pol mutants that retain catalytic activity but are unable to complex with UL42 are incapable of supporting viral replication (Digard et al, 1993a) or origin-dependent DNA synthesis (Stow, 1993).…”

Section: R K Hamatake and Othersmentioning

confidence: 97%

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The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain

Hamatake

Bifano

Tenney

et al. 1993

Journal of General Virology

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Herpes simplex virus type 1 encodes its own DNA polymerase (Pol), the product of the UL30 gene, and a polymerase accessory subunit, the product of the UL42 gene, both of which are required for viral DNA replication. Pol and the UL42 protein associate to form a heterodimeric complex (Pol/UL42) which is more active and has a higher processivity than the Pol catalytic subunit alone. The Pol/UL42 complex has been reconstituted by mixing together highly purified Pol and UL42 subunits obtained from recombinant baculovirusinfected cells. We have used polymerase activity on poly(dA):oligo(dT20), a template that the Pol subunit utilizes with low efficiency, to measure the formation of the Pol/UL42 complex. Our data indicate that the association constant for the Pol/UL42 complex is 1 × l0 s M -1. Proteolytic digestions of UL42 were performed to determine whether structural domains of UL42 could be disclosed by differential amino acid accessibilities. The ability of these protease-resistant domains to form a functional complex with Pol was determined by measuring their ability to stimulate Pol activity on poly(dA):oligo(dT20). We have found that trypsin digestion of UL42 in the presence of DNA generates protease-resistant fragments of 28K and 8K which co-elute from a MonoQ column and are able to stimulate Pol activity on poly(dA): oligo(dT20 ). Complex formation of the 28K and 8K tryptic fragments with Pol was also shown by their co-immunoprecipitation with antibody to Pol. It was determined that the 28K fragment of UL42 comprised amino acids 1 to 245 or 1 to 254 of UL42, whereas the 8K fragment started at amino acid 255. Thus, controlled proteolysis of UL42 revealed two closely contiguous structural domains that retained the ability to complex with Pol and stimulate Pol activity.

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Section: R K Hamatake and Othersmentioning

confidence: 92%

Section: R K Hamatake and Othersmentioning

confidence: 97%

The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain

Hamatake

Bifano

Tenney

et al. 1993

Journal of General Virology

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“…A virus with a deletion of the C-terminal 150 amino acids of the HSV-1 polymerase accessory subunit UL42 displays no obvious defect in replication (9). Thus, it appears that HSV-1 and HCMV exhibit different requirements for the C-terminal segments of their respective accessory proteins.…”

Section: Figmentioning

confidence: 99%

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

Silva

Loregian

Pari

et al. 2010

J Virol

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The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxyterminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.

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“…The UL42 preparation was a C-terminal truncation mutant that retains all known biochemical and biological activities of the full-length protein (20)(21)(22). In contrast to the studies in refs.…”

Section: Effects Of Salt Concentration On Diffusion Calculated From Ementioning

confidence: 99%

Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

Komazin-Meredith

Mirchev

Golan

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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...

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The C-Terminal Third of UL42, a HSV-1 DNA Replication Protein, Is Dispensable for Viral Growth

Cited by 28 publications

References 0 publications

The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain

The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

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