Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.

Jesper Aagaard Christensen; S F Cotmore; P Tattersall

Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.

Jesper Aagaard Christensen, S F Cotmore, P Tattersall

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

44 Citationer (Scopus)

Abstract

Udgivelsesdato: 1997-Aug

Originalsprog	Engelsk
Tidsskrift	Journal of Virology
Vol/bind	71
Udgave nummer	8
Sider (fra-til)	5733-41
Antal sider	8
ISSN	0022-538X
Status	Udgivet - 1997
Udgivet eksternt	Ja

Bibliografisk note

Keywords: Animals; Base Sequence; Binding Sites; DNA; DNA-Binding Proteins; Hela Cells; Humans; Minute virus of mice; Molecular Sequence Data; Molecular Weight; Oligonucleotides; Spodoptera; Viral Nonstructural Proteins

Citationsformater

@article{3c432a60540411dd8d9f000ea68e967b,

title = "Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.",

abstract = "A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.",

author = "Christensen, {Jesper Aagaard} and Cotmore, {S F} and P Tattersall",

note = "Keywords: Animals; Base Sequence; Binding Sites; DNA; DNA-Binding Proteins; Hela Cells; Humans; Minute virus of mice; Molecular Sequence Data; Molecular Weight; Oligonucleotides; Spodoptera; Viral Nonstructural Proteins",

year = "1997",

language = "English",

volume = "71",

pages = "5733--41",

journal = "Journal of Virology",

issn = "0022-538X",

publisher = "American Society for Microbiology",

number = "8",

}

TY - JOUR

T1 - Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.

AU - Christensen, Jesper Aagaard

AU - Cotmore, S F

AU - Tattersall, P

N1 - Keywords: Animals; Base Sequence; Binding Sites; DNA; DNA-Binding Proteins; Hela Cells; Humans; Minute virus of mice; Molecular Sequence Data; Molecular Weight; Oligonucleotides; Spodoptera; Viral Nonstructural Proteins

PY - 1997

Y1 - 1997

N2 - A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.

AB - A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.

M3 - Journal article

C2 - 9223459

SN - 0022-538X

VL - 71

SP - 5733

EP - 5741

JO - Journal of Virology

JF - Journal of Virology

IS - 8

ER -