Analysis of in Vitro Activities of Herpes Simplex Virus Type 1 UL42 Mutant Proteins: Correlation with in Vivo Function

Thornton, Keith; Chaudhuri, Murari; Monahan, Steven J.; Grinstead, Lynn A.; Parris, Deborah S.

doi:10.1006/viro.2000.0506

Cited by 16 publications

(10 citation statements)

References 39 publications

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“…By contrast, the Pol/UL42 holoenzyme activity was relatively resistant to changes in ionic strength over KCl concentrations ranging from 50 to 125 mM. Similar salt resistance was observed for Pol/UL42 holoenzyme reconstituted from the individual subunits (39).…”

Section: Detection Of Binding To P/t By Nitrocellulose Filter Assayssupporting

confidence: 59%

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Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

Chaudhuri

Parris

2002

J Virol

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The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high-versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.The DNA polymerase of herpes simplex virus type 1 (HSV-1) is a heterodimer composed of a catalytic subunit (Pol), encoded by the UL30 gene, and a double-stranded (ds) DNA binding protein, encoded by the UL42 gene (6,13,15,31). The UL42 protein stimulates the activity of Pol on nicked and/or gapped DNA templates in high-salt buffer and increases the processivity of Pol on singly primed single-stranded (ss) DNA templates (12,15,17). The results of biochemical and genetic analyses have revealed that both Pol and UL42 protein are required for extensive leading and lagging strand DNA synthesis in cell-free (in vitro) assays (10,25,35,36) and for viral DNA replication in infected cells (in vivo) (20,22,27,33). The carboxyl-terminal (C-terminal) 227-amino-acid domain of Pol is suffici...

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Section: Detection Of Binding To P/t By Nitrocellulose Filter Assayssupporting

confidence: 59%

“…6). Control experiments demonstrated that BSA at concentrations as high as 600 nM failed to bind to surfaces containing the model P/T at a level significantly higher than to non-DNA-containing surfaces (39).…”

Section: Vol 76 2002mentioning

confidence: 99%

Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

Chaudhuri

Parris

2002

J Virol

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“…Although we do not understand why such a large fraction of the R182A protein in crude extracts was active, these observations illustrate the necessity of first removing inactive protein prior to assaying the binding of UL42 proteins to DNA or to other proteins, including Pol. This issue may be relevant to a report of poor correlations between the effects of mutations on certain in vitro activities of mutant UL42 proteins and their effects in infected cells (31).…”

Section: Discussionmentioning

confidence: 99%

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

Randell

Komazin

Jiang

et al. 2005

J Virol

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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...

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“…43,44 A priori, therefore, one might have expected a gradual increase in processivity as the amount of DNA with which UL42 can interact increases. However, each mutation reduced UL42 binding much more than they reduced the processivity of the UL30-UL42 complex.…”

Section: Discussionmentioning

confidence: 99%

Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase

Dickerson

Kuchta

2017

Biochemistry

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The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42, as well as two non-cognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner incompetent for coupled helicase-polymerase activity. When present, UL42 increases the processivity of UL30 ~ 8-fold and makes a critical interaction with the DNA 24 nucleotides behind the primer 3′-terminus.

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Analysis of in Vitro Activities of Herpes Simplex Virus Type 1 UL42 Mutant Proteins: Correlation with in Vivo Function

Cited by 16 publications

References 39 publications

Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

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

Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase

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