Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently we described the use of human cytomegalovirus (HCMV) cosmid clones in a cotransfection assay of HCMV oriLyt replication (G. S. Pari, M. A. Kacica, and D. G. Anders, J. Virol. 67:2575-2582, 1993). We have now used this assay to identify 11 distinct required loci encoding trans-acting factors sufficient for transient complementation of oriLyt-dependent DNA replication. This set includes all of the virus genes essential to initiate and perform DNA synthesis together with the virus genes required to express these replication functions from their native promoters. Six of the identified loci span open reading frames (ORFs) that encode homologs or probable homologs of herpes simplex virus type 1 replication genes, consistent with predictions based on sequence similarities and biochemical properties. These include the DNA polymerase UL54 and polymerase-associated protein UL44, the single-stranded-DNA-binding protein UL57, and proposed subunits of a helicase-primase complex, UL70, UL105, and UL101-102. Frameshift mutations in any one of these essential ORFs abrogated complementation of DNA replication. Three required loci, UL36-38, IRS1 (or TRS1), and IE1/IE2, encode known regulatory proteins. The remaining two loci span ORFs UL84 and UL112-113 and encode early temporal class nucleus-associated proteins of unknown function. Neither of these genes have been implicated previously in DNA replication or in regulating gene expression, nor have counterparts in herpes simplex virus type 1 or Epstein-Barr virus been described. The results presented here will facilitate investigation of the mechanisms and regulation of HCMV lytic-phase DNA replication.
 ...
PMID:Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication. 823 Apr 21

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.
 ...
PMID:Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. 838 66

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.
 ...
PMID:Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection. 852 51

To study the relationship of the polymorphism of the insulin receptor substrate-1 (IRS-1) gene 5'-flanking regulatory sequence and Type 2 diabetes, the IRS-1 gene 5'-flanking regulatory sequence was scanned by PCR-SSCP in 78 healthy control subjects and 76 Type 2 diabetic subjects. Applying PCR-denatured polyacrylamide gel electrophoresis and silver staining, the insertion/deletion polymorphism of the CAG-rich region was analyzed. The genome DNA of the normal and variant subjects was amplified with high-fidelity pfu DNA polymerase. The purified and digested target fragments were then subcloned into the pCAT Basic vector. Each allele was identified according to the mobility by the restrictive endonuclease digestion of the recombinant combined with denatured polyacrylamide gel electrophoresis and silver staining, and finally the constructive plasmids containing different alleles were analyzed by DNA sequencing. Firstly, we found several insertion/deletion variations in the CAG-rich region of IRS-1 gene. Secondly, 7 genotypes and 6 alleles(T1-T6) in this site were detected. Moreover, T5 and T6 were only observed in Type 2 diabetic group.
 ...
PMID:[Study on base insertion/deletion of CAG-rich region in insulin receptor substrate-1 5'-regulatory sequence]. 1253 35

Multiple proteins interacting with DNA polymerases orchestrate DNA replication. Human cytomegalovirus (HCMV) encodes a DNA polymerase that includes the presumptive processivity factor UL44. UL44 is structurally homologous to the eukaryotic DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous proteins. Previous proteomic analysis has identified the HCMV protein IRS1 as a candidate protein interacting with UL44. Nuclease-resistant reciprocal co-immunoprecipitation of UL44 with IRS1 and with TRS1, which has an amino terminus identical to that of IRS1, was observed from lysate of cells infected with viruses expressing epitope-tagged UL44, epitope-tagged IRS1 or epitope-tagged TRS1. Western blotting of protein immunoprecipitated from infected cell lysate indicated that epitope-tagged IRS1 and TRS1 do not associate simultaneously with UL44. Glutathione S-transferase pull-down experiments indicated that IRS1 and TRS1 interact with UL44 via a region that is identical in both proteins. Taken together, these data suggest that IRS1 and TRS1 may compete for association with UL44 and may affect UL44 function differentially.
 ...
PMID:Association of human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44. 2044 96

Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.
 ...
PMID:A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization. 2285 86