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

The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymerase but fails to stimulate the DNA polymerase activity. The mature p43 is synthesised as a pre-p43 protein containing a 59 amino acid long transit peptide which undergoes stromal cleavage as evidenced from the post-translational in vitro import of the precursor protein into the isolated intact pea chloroplasts. Surprisingly, p43 is found only in pea chloroplasts. The unique features present in the cloned cDNA indicate that p43 is a novel member of the HRGP family of proteins. Besides p43, no other DNA-polymerase accessory protein with O-glycosylation has been reported yet.
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PMID:Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase. 1045 8

p43, a glycoprotein of pea chloroplast (ct), acts as an accessory protein of pea chloroplast DNA polymerase. p43 binds to DNA, binds to ct-DNA polymerase and stimulates the ct-DNA polymerase activity. In the work presented here, the C-terminal domain of p43 (p22) has been overexpressed in E. coli. South Western analysis reveals that the recombinant p22 lacks in DNA binding activity. However, the recombinant p22 can form complex with the pea ct-DNA polymerase quite efficiently and stimulates the DNA polymerase activity to a greater extent than the native p43. Thus the DNA binding domain of p43 appears to be spatially separate from the domain responsible for the DNA polymerase accessory activity. The DNA binding domain is also highly O-glycosylated and loss of glycosylation of p43 leads to enhanced DNA binding as well as repression of ct-DNA polymerase activity. These findings allow us to propose a model to explain how glycosylation of p43 helps ct-DNA polymerase latch onto the DNA template for enhanced processivity. The predictive components of the model have been discussed.
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PMID:Carboxy terminal region of a chloroplast DNA polymerase accessory factor stimulates DNA polymerase activity. 1135 29

The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.
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PMID:Role of the specific interaction of UL112-113 p84 with UL44 DNA polymerase processivity factor in promoting DNA replication of human cytomegalovirus. 2053 62

The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle.IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene products was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.
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PMID:Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication. 2863 62