Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

Dornreiter, I; Copeland, William C.; Wang, T S

doi:10.1128/mcb.13.2.809

Cited by 99 publications

(121 citation statements)

References 31 publications

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“…This interpretation is supported by the earlier finding that the addition of the Tag binding site within the N terminus of p180/DNA polymerase ␣ inhibited SV40 DNA replication in vitro only during the so-called lag period of the assay. Later, when the leading and lagging strand are replicated and the priming of the Okazaki fragments on the lagging strand is the only task of the primase, the addition of the polypeptide could no longer inhibit or reduce the incorporation of dNMPs (30). Our interpretation, that the initiation reactions on the leading and lagging strand require in part dissimilar protein-protein interactions and that the mechanisms of both processes might differ (Fig.…”

Section: Figmentioning

confidence: 71%

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: 71%

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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“…1A). Interestingly, the residues 195-313 of human p180, thought to bind SV40 T antigen (Dornreiter et al, 1993), and the corresponding residues 201-319 of mouse pl80 protein were the least conserved (only 66% identical, Fig. 1 A).…”

Section: Resultsmentioning

confidence: 99%

“…1A). Some of the most highly conserved regions are involved in the catalytic activity of DNA polymerase a (Copeland and Wang, 1993).…”

Section: Resultsmentioning

confidence: 99%

DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase

Stadlbauer

Brueckner

Rehfuess

et al. 1994

European Journal of Biochemistry

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DNA-polymerase-a-primase complex contains four subunits, pl80, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-a-primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNApolymerase-a-primase. The pl80, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-aprimase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-a-primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.The study of enzymic mechanisms, fidelity and regulation of DNA replication is fundamental to a detailed understanding of the stability of genetic information. DNA replication of eukaryotic genomes can be divided into several steps; recognition of an origin of DNA replication, assembly of a pre-initiation complex, unwinding of origin DNA, initiation of replication within an origin and elongation of leading and lagging strands (Kornberg and Baker, 1991). DNA polymerase a and DNA primase are essential components of the cellular DNA replication machinery (Pizzagalli et al., 1988 ; Francesconi et al., 1991), playing an essential role both in initiation and in lagging strand synthesis (Thommes and Hubscher, 1990;Kornberg and Baker, 1991 ;.DNA primase and DNA polymerase a co-purify as a complex containing four subunits with molecular masses of 180, 68, 58 and 48kDa (Thommes and Hubscher, 1990;. The p380 subunit of DNA-polymerase-a-primase carries the DNA polymerase activity, and is phosphorylated in a cell-cycle-dependent manner (Pizzagalli et al.,

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“…Immunological Methods-Polyclonal antibody ID␣, raised in chicken against the recombinant p180 subunit of human DNA polymerase ␣, was kindly provided by Teresa Wang (Stanford University, Stanford, CA) (36). Antiserum to DNA polymerase ␦ was raised in a New Zealand White rabbit against the synthetic multiple antigenic peptide LYQKEVSHLSALEERFSRLW, corresponding to residues 1036 -1055 of the catalytic subunit of calf thymus DNA polymerase ␦ (37).…”

Section: Preparation Of Nuclear Extracts and A Depleted Extract Deficmentioning

confidence: 99%

DNA Polymerase δ Is Required for Human Mismatch Repair in Vitro

Longley

Pierce

Modrich

1997

Journal of Biological Chemistry

200

138

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HeLa nuclear extract was resolved into a depleted fraction incapable of supporting mismatch repair in vitro, and repair activity was restored upon the addition of a purified fraction isolated from HeLa cells by in vitro complementation assay. The highly enriched complementing activity copurified with a DNA polymerase, and the most pure fraction contained DNA polymerase ␦ but was free of detectable DNA polymerases ␣ and ⑀. Calf thymus DNA polymerase ␦ also fully restored mismatch repair to the depleted extract, indicating DNA polymerase ␦ is required for mismatch repair in human cells. However, due to the presence of DNA polymerases ␣ and ⑀ in the depleted extract, potential involvement of one or both of these activities in the reaction cannot be excluded.Mismatch repair ensures genetic stability by correcting DNA biosynthetic errors and by blocking recombination between diverged DNA sequences (reviewed in Refs. 1-3). Defects in human MutS␣ or MutL␣ result in genetic destabilization (4 -8), are the genetic basis of tumor predisposition in hereditary nonpolyposis colorectal cancer kindreds (9 -13), and have been implicated in a subset of sporadic cancers (5, 14 -17). In addition to its ability to recognize mismatched base pairs (16, 18), MutS␣ also recognizes certain classes of DNA damage (19,20), and MutS␣ or MutL␣ defects are associated with resistance to certain DNA-damaging agents, including several that are used in anticancer chemotherapy (4,(21)(22)(23)(24)(25)(26).The Escherichia coli mismatch repair reaction has been reconstituted in vitro (27,28). Briefly, MutS recognizes a mismatch and MutL binds to this complex. This activates a latent, MutH-associated endonuclease that incises the unmethylated strand of a hemimethylated d(GATC) sequence, which may be located either 5Ј or 3Ј to the mispair. The nick serves as the primary signal that directs repair to the unmethylated strand, and in the presence of DNA helicase II, exonucleolytic excision initiates at the nick and proceeds toward the mismatch through the action of either RecJ or exonuclease VII when incision is 5Ј to the mismatch, or exonuclease I when the nick is 3Ј to the mispair (28, 29). The resulting single-strand gap is then filled by DNA polymerase III holoenzyme, and covalent continuity is restored to the repaired strand by DNA ligase. Single-stranded DNA-binding protein is also a required component of the reaction.Four activities have been implicated in the human mismatch repair reaction. MutS␣ (a heterodimer of MSH2 and MSH6) and MutL␣ (a heterodimer of MLH1 and PMS2) were isolated by virtue of their ability to restore strand-specific mismatch correction to nuclear extracts of repair-deficient tumor cell lines (16,18,30). In addition to its subunit function in MutS␣, MSH2 forms a molecular complex with the MutS homolog MSH3 (31, 32). This MutS␤ complex binds to insertion/deletion mismatches and presumably plays a role in their processing. Proliferating cell nuclear antigen (PCNA) 1 has also been implicated in the human mismatch repair reaction ...

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Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

Cited by 99 publications

References 31 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

DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase

DNA Polymerase δ Is Required for Human Mismatch Repair in Vitro

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