Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

Murakami, Yasufumi; Wobbe, C R; Weissbach, Lawrence; Dean, Frank B.; Hurwitz, Jerard

doi:10.1073/pnas.83.9.2869

Cited by 218 publications

(159 citation statements)

References 22 publications

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“…Therefore, mouse RPA is not a species-specific factor and is not the source of the inhibitory activities. Similar to RPA, Pol-prim is essential for eukaryotic DNA replication and is the major species-specific factor of mPyV and SV40 (8,32,33,50). Here, we verified the ability of mouse Pol-prim to functionally and physically interact with BKV TAg, similar to its interaction with SV40 TAg in the SV40 system (10,53,57).…”

Section: Discussionsupporting

confidence: 52%

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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Section: Discussionsupporting

confidence: 52%

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 this case, RNA priming is essential for lagging-strand synthesis. Priming in eukaryotes is carried out by the primase activity of the RNA-primase-Pol-α complex 40,41 and is essential for lagging-strand synthesis, as well as for the initiation of leading-strand synthesis in normal DNA replication.Once the RNA primer is synthesized, it is extended by the Pol α generating a short DNA primer onto which the Pol δ system is assembled, similar to the leading-strand synthesis step (Fig. 2).…”

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confidence: 99%

Links between replication, recombination and genome instability in eukaryotes

Flores‐Rozas

Kolodner

2000

Trends in Biochemical Sciences

107

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Double-strand breaks in DNA can be repaired by homologous recombination including breakinduced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.The ability of cells to repair double-strand breaks (DSBs) is critical for cell viability. Failure to correct such damage can result in cell cycle arrest, cell death and, if repaired incorrectly, the loss of genetic information or the accumulation of mutations. DSBs can arise in cells by exposure to ionizing radiation 1 and other types of DNA-damaging agents or through mechanical stress. In addition, some cellular processes, like DNA replication, can generate DSBs. These could arise by the action of nucleases at unprotected sites where replication forks stall, as a consequence of DNA polymerases (Pols) replicating across nicked chromosomes, as well as by the active processing of stalled replication forks by specific enzyme systems [2][3][4] . DSBs can also occur naturally and play important roles in processes such as meiosis 5 , mating-type switching 6 and mammalian V(D)J recombination 7 .Cells have developed a number of mechanisms to repair such lesions. In Saccharomyces cerevisiae, most of the repair of DSBs is carried out by mechanisms that promote homologous recombination. This requires the existence in the cell of sequences that are homologous to those affected by the DSB. Non-homologous end-joining also rejoins DSBs in S. cerevisiae albeit less efficiently than homologous recombination; non-homologous endjoining appears to be of greater importance in mammalian cells 8 . General models of recombinationSeveral recent reviews have discussed recombination and the repair of DSBs in detail 4,9-14 . Given the increasing evidence that translocations and other types of genome rearrangements arise from inappropriate repair of DSBs, we will focus on the repair of DSBs by a homologous recombination pathway that involves extensive DNA synthesis. Such a process, termed break-induced replication (BIR), has been suggested based on the observations by 16 . In this study, activation of the transcription enhancer element HOT1 creates a recombination hotspot suggested to act by generating DSBs that are processed resulting in strains with distal markers converted to those present in the homologous chromosome. The resulting gene conversion, which could extend over regions as long as 75 kb, was proposed to arise by replication of a primer structure generated by the centromere-containing fragment invading the homologous chromosome in a process analogous to that described for bacteriophage T4 (Ref. 17 (Fig. 1a). In both cases the ends of the broken DNA molecule are degraded by exonucleases creating 3′ overhangs that invade the homologous template. The result is the formation of a D-loop in which the annealed invading end serves as a primer that is extended by the DNA syn...

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“…Whether this is a common factor in determination of species specificity of polyomaviruses in general remains to be seen, but the similarity in replication mechanisms throughout this virus family would make it a likely possibility. Species specificity of replication can be reproduced in cell-free systems, and this led to the demonstration that DNA polymerase ␣-primase is the host factor that determines the species specificity of viral initiation of DNA replication of both SV40 and PyV (25)(26)(27). However, studies with hybrid DNA polymerase ␣-primase complexes showed that the subunit requirement differs between these two viruses, PyV specifically needing p48 to be of murine origin and SV40 requiring a human p180 protein (28,29).…”

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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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Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

Cited by 218 publications

References 22 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

Links between replication, recombination and genome instability in eukaryotes

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

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