scholarly journals The 3‘-5‘ proofreading exonuclease of bacteriophage T4 DNA polymerase is stimulated by other T4 DNA replication proteins.

1983 ◽  
Vol 258 (16) ◽  
pp. 9649-9656 ◽  
Author(s):  
P Bedinger ◽  
B M Alberts
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Replication Proteins

scholarly journals Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

2010 ◽  
Vol 84 (17) ◽  
pp. 8409-8421 ◽  
Author(s):  
Young-Eui Kim ◽  
Jin-Hyun Ahn
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Specific Interaction ◽  
Terminal Region ◽  
Replication Proteins ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Carboxy Terminal Region ◽  
Processivity Factor ◽  
Carboxy Terminal

ABSTRACT 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.

scholarly journals DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1739
Author(s):  
Chen-Yu Lo ◽  
Yang Gao
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Molecular Mechanisms ◽  
Bacteriophage T4 ◽  
Dna Helicase ◽  
Model Systems ◽  
Genome Replication ◽  
Template Strand ◽  
Replication Systems ◽  
Mechanistic Basis

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

[43] Purification of bacteriophage T4 DNA replication proteins

1995 ◽  
pp. 560-584 ◽  
Author(s):  
Nancy G. Nossal ◽  
Deborah M. Hinton ◽  
Lisa J. Hobbs ◽  
Peter Spacciapoli
Keyword(s):  
Dna Replication ◽  
Bacteriophage T4 ◽  
Replication Proteins

scholarly journals Changes in Organization of Crithidia fasciculata Kinetoplast DNA Replication Proteins during the Cell Cycle

1998 ◽  
Vol 143 (4) ◽  
pp. 911-919 ◽  
Author(s):  
Catharine E. Johnson ◽  
Paul T. Englund
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Topoisomerase Ii ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Dna Polymerase Β ◽  
Cycle Dependent

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle–dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein–dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase β colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase β is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

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

2009 ◽  
Vol 83 (15) ◽  
pp. 7581-7589 ◽  
Author(s):  
Blair L. Strang ◽  
Elisa Sinigalia ◽  
Laurie A. Silva ◽  
Donald M. Coen ◽  
Arianna Loregian
Keyword(s):  
Dna Replication ◽  
Infected Cell ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Nuclear Antigen ◽  
Replication Proteins ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Processivity Factor ◽  
Replication Factors

ABSTRACT 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.

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