The Human Cytomegalovirus UL44 C Clamp Wraps around DNA

Komazin-Meredith, Gloria; Petrella, Robert J.; Santos, Webster L.; Filman, David J.; Hogle, James M.; Verdine, Gregory L.; Karplus, Martin; Coen, Donald M.

doi:10.1016/j.str.2008.05.008

Cited by 30 publications

(36 citation statements)

References 56 publications

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“…It should be interesting to elucidate these mechanisms. Finally, although UL44 and PCNA are structurally homologous (2), the mechanism of UL44 binding to DNA (14), the mechanism of UL54 binding to UL44 (1), and the results we present here indicate notable evolutionary divergence between UL44 and PCNA. This observation leads, in turn, to the suggestion that there may be more as-yet-unknown functions of UL44 and PCNA that the two proteins do not share.…”

Section: Discussionmentioning

confidence: 62%

“…UL44 interacts with UL54 and DNA and stimulates long-chain DNA synthesis (21,39) and thus very likely serves as a processivity factor. UL44 forms a head-to-head homodimer (1) that binds DNA without the aid of clamp loaders yet wraps around DNA akin to PCNA (14). While UL44 shows little or no sequence homology with PCNA, there is striking structural similarity between UL44 and PCNA monomers (1,2).…”

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confidence: 99%

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Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

Strang

Sinigalia

Silva

et al. 2009

J Virol

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The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-S-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.Protein-protein interactions orchestrate the process of DNA synthesis. Most replicative DNA polymerases include at least two interacting components, a catalytic subunit responsible for the polymerization of DNA and a processivity subunit. The processivity subunit of the polymerase holds the catalytic subunit on DNA while DNA polymerization takes place, thereby permitting long-chain DNA synthesis. One of the best-described processivity factors is proliferating cell nuclear antigen (PCNA) of eukaryotic DNA polymerases ␦ and ε (23, 26). PCNA is a head-to-tail homotrimer which, with the aid of clamp loader proteins, forms a toroidal ring around DNA, creating an internal channel of sufficient diameter to accommodate the DNA duplex (15). Studies of PCNA by numerous laboratories have described a large number of interactions between PCNA and proteins that participate in and abet DNA synthesis (reviewed in references 23 and 26). Of particular interest is that many of these proteins bind PCNA at a specific site, inserting a conserved hydrophobic domain (a PCNA-interacting protein [PIP] box) into a hydrophobic pocket lying beneath an interdomain connector loop of PCNA. PIP box proteins bind and dissociate from PCNA at different times during DNA replication when the need for their funct...

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Section: Discussionmentioning

confidence: 62%

mentioning

confidence: 99%

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

Strang

Sinigalia

Silva

et al. 2009

J Virol

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“…BMRF1 Binds dsDNA on Its Concave Surface as a DimerThe crystal structure of the E. coli polymerase ␤-subunit revealed that dsDNA binds to the inside of the ring structure, through basic amino acid residues (44). Although there is no complex structure between DNA and a virus polymerase processivity factor available thus far, site-directed mutation analyses of HSV-1 UL42 supported the proposal that protein-DNA interactions occur with the positively charged amino acid residues on the "back" face (40).…”

Section: Discussionmentioning

confidence: 99%

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

Murayama

Nakayama

Kato-Murayama

et al. 2009

Journal of Biological Chemistry

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The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-⌬C) was solved in an oligomeric state. The molecular structure of BMRF1-⌬C shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-⌬C, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-⌬C exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-⌬C associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-⌬C probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-⌬C is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.The Epstein-Barr virus (EBV), 4 a human herpesvirus harboring a 172-kbp dsDNA genome, is associated with several B-cell and epithelial cell malignancies and can choose between two alternative life cycles, latent and lytic infection (1). The EBV genomes are replicated as circular plasmid molecules, using the cellular replication machinery of the host in the latent phase of the viral life cycle. On the other hand, after the induction of lytic viral replication, the EBV genome is amplified 100 -1,000-fold by the viral replication machinery. The replication intermediates are large head-to-tail concatemers resulting from rolling-circle DNA replication initiated from oriLyt (2). The EBV replication machinery consists of seven viral gene products (3) as follows: the BZLF1 protein, an oriLytbinding protein; the BALF5 protein, a DNA polymerase catalytic subunit; the BMRF1 protein, a polymerase processivity factor; the BALF2 protein, a single-stranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, putative helicase, primase, and helicase-primase-associated proteins, respectively. It has been suggested that all of the proteins, except for the BZLF1 protein, work together at replication forks to synthesize the leading and lagging strands of the concatemeric EBV genome (2). The EBV DNA polymerase holoenzy...

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“…Thus, it appears that HSV-1 and HCMV exhibit different requirements for the C-terminal segments of their respective accessory proteins. This and many other differences between these functionally and structurally orthologous proteins (5,6,20,24,25) suggest considerable selection for different features during evolution.…”

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 Human Cytomegalovirus UL44 C Clamp Wraps around DNA

Cited by 30 publications

References 56 publications

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

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

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

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