Different Regions of Primase Subunit p48 Control Mouse Polyomavirus and Simian Virus 40 DNA Replication In Vitro

Kautz, Armin R.; Schneider, A; Weißhart, Klaus; Geiger, Christiane; Nasheuer, Heinz-Peter

doi:10.1128/jvi.75.4.1751-1760.2001

Cited by 9 publications

(21 citation statements)

References 52 publications

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“…It is known that SV40 DNA replication requires only the presence of the large p180 DNA polymerase subunit of human origin in a chimeric Pol-prim complex with the three small subunits of mouse origin to replicate SV40 DNA in mouse extracts in the presence of SV40 TAg (50). In contrast, Pol-prim with the small p48 primase subunit of mouse origin and the three other subunits of human origin is sufficient to allow mPyV DNA replication in human cell extracts in the presence of PyV TAg (8,23). Since Pol-prim is an important species-specific factor for BKV replication as well, we studied how different mousehuman hybrid Pol-prim complexes containing subunits of both human and mouse origin support BKV DNA replication.…”

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confidence: 94%

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Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

Tikhanovich

Nasheuer

2010

J Virol

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The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase ␣-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

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confidence: 94%

“…on May 9, 2018 by guest http://jvi.asm.org/ in human and mouse cell extracts, respectively (8,23,46,50). The activity of human and mouse Pol-prim was tested in assays with ssDNA as a template ( Fig.…”

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confidence: 99%

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

Tikhanovich

Nasheuer

2010

J Virol

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“…In the case of PyV, further resolution was achieved with studies of DNA polymerase ␣-primase complexes containing chimeric p48 subunits consisting of various combinations of human and murine sequences. It was concluded that a lesser conserved stretch of amino acids (residues 257-288) from the murine subunit was necessary for successful PyV DNA replication, and it is likely that further study of the function of this region will shed light on the mechanism involved in this phenomenon (30,31). In this study we applied a similar approach in an attempt to determine which domains of human p180 are essential for initiation of SV40 DNA replication.…”

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confidence: 99%

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Smith¹,

Steffen²,

Grosse³

et al. 2002

Journal of Biological Chemistry

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Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase ␣-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase ␣-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase ␣-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase ␣ depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase ␣-primase but rather on sequences in the C-terminal region of human p180.Polyomavirus DNA replication has been studied extensively because of the ease with which viruses of this family, which include SV40 and mouse polyoma virus (PyV), 1 can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed investigation of the individual factors that play a role in the replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the host DNA but does contribute one essential trans-acting factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at which it forms a double hexamer (3). In the presence of the single-stranded DNA-binding protein, RPA (replication protein A), and topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the DNA polymerase ␣-primase heterotetramer, which then initiates bidirectional DNA synthesis (4 -11). DNA polymerase ␣-primase initiates DNA replication through the action of its smallest subunit, p48, which synthesizes RNA primers that are subsequently elongated by the large subunit, p180 (12-15). The bulk of DNA synthesis is, however, carried out by a more processive enzyme complex consisting of DNA polymerase ␦ and its processivity factor, PCNA (16 -18). The transition between DNA synthesis by DNA polymerases ␣ and ␦ is mediated by the PCNA loading factor replication factor C (16, 19). Throughout the viral replication cycle, TAg double hexamers are thought to act as the replicative helicase (20). For a more extensive account of the DNA replication process and its participating factors see Refs. 1, 2, and 21-23. SV40 and PyV are very similar with regard to DNA replication; their respectiv...

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“…The murine p48 subunit and specific regions therein control the replication of PyV DNA in vitro (6,18,31). The catalytic subunit p48 of primase is highly conserved between mice and humans (Ͼ90% identical amino acids).…”

Section: Resultsmentioning

confidence: 99%

“…This species specificity can be reproduced in cell-free DNA replication systems, and the cellular pol-prim is the major species-specific factor for PyV and SV40 DNA replication (6,18,40,42,58). Recently, it was shown that a central region of murine p48 is required for the initiation and elongation steps of PyV DNA replication (31). To determine the cellular requirements for the host-specific DNA replication of PyV and whether one or more regions of p48 are involved in the regulation of PyV DNA replication, chimerical polypeptides were created by exchanging various stretches of mouse and human p48.…”

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Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

Kautz¹,

Weißhart²,

Schneider³

et al. 2001

J Virol

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Although p48 is the most conserved subunit of mammalian DNA polymerase ␣-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein.Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.The small DNA tumor viruses simian virus 40 (SV40) and mouse polyomavirus (PyV) have provided excellent model systems to study the mechanisms and regulation of eukaryotic DNA replication (reviewed in references 7, 9, 55, and 67). Their DNA replication in vivo and in vitro largely depends on the replication apparatus of their hosts, as only one protein, namely, the multifunctional viral large T antigen (Tag), is supplied by the virus (20,26,49).The establishment of cell-free SV40 and PyV DNA replication systems was a major step towards the purification and characterization of essential host replication factors (67). These studies allowed the understanding of the molecular mechanisms involved in the assembly and progression of replication forks (7,28,67). Along with genetic and other biochemical approaches, these DNA replication systems provided insights into the processes of initiation and elongation (67). Interestingly, several parallels between eukaryotic and prokaryotic DNA duplication emerged which show that central processes of DNA metabolism are in part conserved in all kingdoms (33,60,61). The studies of the polyomaviral systems have allowed the development of a general model for eukaryotic DNA replication. In an initial step, Tag recognizes the replication origin (ori) and forms a double hexameric complex, which causes specific distortions of the double-stranded (ds) DNA (4,15,20,37,49). Subsequently, dsDNA is unwound, and multiprotein complexes are assembled by Tag and host replication proteins, such as eukaryotic single-stranded (ss) DNA-binding protein (replication protein A [RPA]), topoisomerase I (topo I), and DNA polymerase ␣-primase (polprim), are recruited (10,11,17,23,38,53,54). After binding and distortion of the dsDNA, the helicase function of Tag melts and unwinds the ds ori DNA (4, 59). For this function, Tag depends on RPA as a cofactor to stabilize the ssDNA resulting from the helicase activity of Tag (7,29,72). Th...

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Different Regions of Primase Subunit p48 Control Mouse Polyomavirus and Simian Virus 40 DNA Replication In Vitro

Cited by 9 publications

References 52 publications

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

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