Progress Towards the Anatomy of the Eukaryotic DNA Replication Fork

Nasheuer, Heinz-Peter; Pospiech, Helmut; Syväoja, Juhani E.

doi:10.1007/7050_016

Cited by 15 publications

(14 citation statements)

References 206 publications

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“…The addition of the auxiliary factor Tag is required that Pol-prim is able to synthesise RNA and DNA on ssDNA in the presence of RPA. The components required for DNA synthesis are shown: ssDNA (single purple line); RPA (in brown the large subunit RPA70 with the OB-fold domains A, B, C, and F; in beige middle subunit RPA32 containing N-terminal phosphorylation sites, the central OB-fold D and the protein interaction region RPA32C, and the small RPA14 subunit in orange consisting of the OB-fold domain E [12,13]); Pol-Prim (four subunits: DNA polymerase [10,56] subunit p180 in dark blue consisting of N-terminal Tag interaction region (p180N) and the catalytic DNA polymerase and additional protein interaction sites, with its core regulatory subunit p68 in blue, consisting of the p68 N-terminal region (p68N) that interacts with SV40 Tag and the remaining p68 residues necessary for DNA replication [48]; and in light blue the two primase subunits p48 (Prim 1) containing the catalytic site and the large primase subunit p58 (Prim2) that interacts with the large subunit p180); and the auxiliary factor Tag (green, showing only the minimal regions required for the stimulation of Pol-prim, the OBD and the helicase domain with aa V350 and P417 necessary for the stimulation shown as V and P and highlighted in red-brown). Interactions between Pol-prim and Tag are indicated: p68N (N) with the AAA+ domain of Tag (Fig.…”

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SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation

et al. 2022

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The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α‐primase (Pol‐prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single‐stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA‐bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol‐prim on free and RPA‐bound ssDNA. Atomic force microscopy imaging showed that while Tag131‐627(V350E/P417D) and Tag131‐627(L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full‐length Tag SV40 Tag1‐708 and monomeric M2 stimulated DNA synthesis of Pol‐prim on ssDNA and on RPA‐bound ssDNA. In contrast, neither monomeric M1 nor M1‐M2 dimers could stimulate Pol‐prim, on ssDNA or on RPA‐bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1‐M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein–protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

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SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation

et al. 2022

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“…Then, DNA polymerase ␣-primase (Polprim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex. In the following step, Pol-prim synthesizes short RNA primers at the origin, and these RNA primers are elongated by the DNA polymerase function of the enzyme complex (9,35,47). After a polymerase switch from Pol-prim to DNA polymerase ␦ (Pol ␦) with the help of RPA, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA), processive DNA synthesis is completed by Pol ␦ in association with PCNA, the sliding clamp, on the leading strand (38,51,54,59).…”

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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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“…and additional levels of control to eukaryotic DNA replication and may lead to the development of new strategies to inhibit replication of viruses or other infections agents. Thus, as for ncRNAs in gene expression (3,13,16,20), srRNAs may contribute to regulation of DNA replication in addition to the well-known factors such as posttranslational modifications, protein-protein interactions, and protein stability (8,27,29,33,34).…”

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Inhibition of Human BK Polyomavirus Replication by Small Noncoding RNAs

Tikhanovich

Liang

Seoighe

et al. 2011

J Virol

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Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.Small noncoding RNAs (ncRNAs) have important roles in eukaryotic gene expression determining developmental processes and growth and differentiation (reviewed in references 1, 8, 20, 27, 30, and 49). In addition, certain ncRNAs (e.g., Y-RNAs) are required for chromosomal DNA replication and proliferation of mammalian cells (5, 23); however, the mechanisms by which these act are not yet understood. Roles for ncRNAs in regulating viral infections of mammalian cells were uncovered with the discovery of virus-and host-encoded RNAs controlling viral gene expression (reviewed in references 10 and 41) and with the recognition that recruitment of the cellular origin recognition complex (ORC) to the viral origin of replication (OriP) by Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) and initiation of EBV DNA replication are RNA dependent (38).BK virus (BKV) infects nearly the entire human population but generally is innocuous unless viral replication is activated following kidney transplantation and immune suppression, when it may cause polyomavirus-associated nephropathy (PVAN) and allograft loss (14,19). Polyomavirus DNA replication in vitro and in vivo requires one viral protein, the large T antigen (TAg), with other factors supplied by the host cell (25, 28). Polyomavirus DNA replication initiates with TAg binding at the viral origin of replication (39, 44) and recruitment of host factors such as replication protein A (RPA), DNA polymerase ␣-primase (Pol-primase), and topoisomerase I (2, 45), followed by Pol-primase catalyzed de novo synthesis of nascent primers for DNA replication. Early initiation products, the nascent DNA primers, are then extended by DNA polymerase ␦ and proliferating cell nuclear antigen (PCNA) and the loading factor replication factor C on the leading and lagging strands (37,54). Although the DNA replication machineries are conserved between primates and rodents, BKV does not...

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Progress Towards the Anatomy of the Eukaryotic DNA Replication Fork

Cited by 15 publications

References 206 publications

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation

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

Inhibition of Human BK Polyomavirus Replication by Small Noncoding RNAs

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