Purification and functional characterization of bovine RP-A in anin vitro SV40 DNA replication system

Nasheuer, Heinz-Peter; Winkler, Dorothea von; Schneider, Christine A.; Dornreiter, Irena; Gilbert, Ilka; Fanning, Ellen

doi:10.1007/bf02451786

Cited by 55 publications

(64 citation statements)

References 44 publications

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“…The failure to detect these interactions in previous reports (28,29,39) might be due to several reasons. In our present investigation, all proteins were from human origin (13, 21, 22, 55, 57).…”

Section: Figmentioning

confidence: 93%

“…Proteins-SV40 T antigen, RPA, the primase (p58-p48, prim 2 ), and the DNA polymerase ␣-primase (pol-prim) complex (p180-p68-p58-p48) were purified from baculovirus-infected insect cells as described (22,28,39,55,56). In addition, RPA, prim 2 , and the individual primase subunits p58 and p48 were bacterially expressed and purified as outlined before (13,57).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, earlier investigations used prim 2 that was purified from non-permissive cells, i.e. from calf thymus tissue or other mammalian organisms (28,29,39). Furthermore, all proteins used in this study were recombinant proteins.…”

Section: Figmentioning

confidence: 98%

“…One site at the N terminus of p70 was responsible for the stimulation of DNA polymerase activity; the other, located within the major DNA binding region of p70, is necessary to increase DNA polymerase processivity (41). However, RPA has been shown to interact with prim 2 and subsequently an interaction with the p48 subunit was recorded (29,39). These reported interactions seem to explain all requirements for the initiation of leading and lagging strand DNA synthesis (1).…”

Section: Figmentioning

confidence: 99%

“…It is believed that these protein-protein interactions are important for the observed stimulation of both the primase and DNA polymerase activity by Tag as well as for the increased origin DNA binding activity of Tag in the presence of pol-prim (18,37,38). Complex formation between the p70 subunit of RPA and the heterodimeric primase consisting of p58 and p48 subunits has been demonstrated, and the p48 subunit on its own seemed to be sufficient for this interaction (29,39). Through these interactions RPA stimulates the polymerase activity and increases the processivity of pol-prim (40 -43).…”

mentioning

confidence: 99%

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Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Weißhart¹,

Förster²,

Kremmer³

et al. 2000

Journal of Biological Chemistry

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DNA polymerase ␣-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim 2 ) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim 2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2⅐10 ؊8 M and 1.3⅐10 ؊8 M, respectively.In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process (reviewed in Ref. 1). For each reaction a specific set of enzymes and accessory proteins is required to replicate chromosomal DNA (1-4). The first step in leading strand DNA replication is accomplished by the synthesis of oligoribonucleotides, called RNA primers, at the origin of DNA replication. This process is carried out by a special enzyme, DNA primase (5-7). The eukaryotic enzyme consists of two subunits, the catalytic subunit p48 and p58, which serves to stabilize the primase activity (8 -13). In eukaryotic cells these two subunits assemble together with the catalytic DNA polymerase subunit, p180, and the p68 polypeptide into a heterotetrameric DNA polymerase ␣-primase complex (pol-prim) 1 (1-7, 14, 15).The initiation of DNA replication requires the interaction of several proteins in vivo and in vitro (1). The start of DNA synthesis de novo occurs by two independent processes: the singular priming event on the leading strand at the origin and the multiple priming steps for Okazaki fragment synthesis on the lagging strand. Both tasks are carried out by the primase activity (1-3, 14 -17). Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded (ss) DNA templates bound by replication protein A (RPA) (17, 18). Using these cell-free systems, it was shown that the initiation of SV40 DNA replication at the origin is species-specific and requires the activity of the p180 subunit of primate pol-prim (19 -21). In contrast to these results, the initiation of Okazaki fragments during lagging strand DNA synthesis is not species-specific and the human as well as the bovine pol-prim can perform lagging strand initiat...

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Section: Figmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 98%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Weißhart¹,

Förster²,

Kremmer³

et al. 2000

Journal of Biological Chemistry

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Replication Protein A Induces the Unwinding of Long Double-stranded DNA Regions

Treuner

Ramsperger

Knippers

1996

Journal of Molecular Biology

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Simian Virus 40 Large T Antigen Binds to Topoisomerase I

et al. 1996

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Binding of simian virus 40 (SV40) large T antigen to human and calf thymus topoisomerase I (topo I) was readily detected by using modified enzyme-linked immunosorbent assays and immunoblots. In addition to WT T antigen, binding could also be readily demonstrated with T antigen fragments from the amino-terminal region as well as with fragments missing this region, but much less so with small t antigen or with human p53. Antibody-blocking experiments showed that a monoclonal antibody that binds to the N-terminal region and several antibodies that recognize the central region of T antigen interfere with the binding to topo I. Our data are consistent with the existence of two separate topo I-binding regions in T antigen, one mapping within residues 82 to 246 and an apparently weaker one present after residue 246. By comparing the binding of T antigen to topo I with that of T antigen to DNA polymerase alpha or RPA, a single-stranded DNA-binding protein, it was determined that the T antigen-topo I interaction is much stronger and that the binding sites for topo I and DNA polymerase overlap, whereas the one for RPA differs. Several unwinding-defective mutants of T antigen were partially defective in their binding to topo I, suggesting that the binding to topo I is required for unwinding circular DNA. Finally, immunoprecipitation experiments demonstrated that T antigen can interact with DNA-bound topo I, indicating that such an interaction may take place during SV40 DNA replication.

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Purification and functional characterization of bovine RP-A in anin vitro SV40 DNA replication system

Cited by 55 publications

References 44 publications

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Replication Protein A Induces the Unwinding of Long Double-stranded DNA Regions

Simian Virus 40 Large T Antigen Binds to Topoisomerase I

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