Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Aug;71(8):5733–5741. doi: 10.1128/jvi.71.8.5733-5741.1997

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

J Christensen 1, S F Cotmore 1, P Tattersall 1
PMCID: PMC191825  PMID: 9223459

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.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Astell C. R., Chow M. B., Ward D. C. Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication. J Virol. 1985 Apr;54(1):171–177. doi: 10.1128/jvi.54.1.171-177.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldauf A. Q., Willwand K., Mumtsidu E., Nüesch J. P., Rommelaere J. Specific initiation of replication at the right-end telomere of the closed species of minute virus of mice replicative-form DNA. J Virol. 1997 Feb;71(2):971–980. doi: 10.1128/jvi.71.2.971-980.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Christensen J., Cotmore S. F., Tattersall P. A novel cellular site-specific DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication. J Virol. 1997 Feb;71(2):1405–1416. doi: 10.1128/jvi.71.2.1405-1416.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christensen J., Cotmore S. F., Tattersall P. Minute virus of mice transcriptional activator protein NS1 binds directly to the transactivation region of the viral P38 promoter in a strictly ATP-dependent manner. J Virol. 1995 Sep;69(9):5422–5430. doi: 10.1128/jvi.69.9.5422-5430.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cotmore S. F., Christensen J., Nüesch J. P., Tattersall P. The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3. J Virol. 1995 Mar;69(3):1652–1660. doi: 10.1128/jvi.69.3.1652-1660.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cotmore S. F., D'Abramo A. M., Jr, Carbonell L. F., Bratton J., Tattersall P. The NS2 polypeptide of parvovirus MVM is required for capsid assembly in murine cells. Virology. 1997 May 12;231(2):267–280. doi: 10.1006/viro.1997.8545. [DOI] [PubMed] [Google Scholar]
  7. Cotmore S. F., Nuesch J. P., Tattersall P. In vitro excision and replication of 5' telomeres of minute virus of mice DNA from cloned palindromic concatemer junctions. Virology. 1992 Sep;190(1):365–377. doi: 10.1016/0042-6822(92)91223-h. [DOI] [PubMed] [Google Scholar]
  8. Cotmore S. F., Nüesch J. P., Tattersall P. Asymmetric resolution of a parvovirus palindrome in vitro. J Virol. 1993 Mar;67(3):1579–1589. doi: 10.1128/jvi.67.3.1579-1589.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cotmore S. F., Tattersall P. An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication. EMBO J. 1994 Sep 1;13(17):4145–4152. doi: 10.1002/j.1460-2075.1994.tb06732.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cotmore S. F., Tattersall P. In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules. J Virol. 1992 Jan;66(1):420–431. doi: 10.1128/jvi.66.1.420-431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  12. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  13. Im D. S., Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell. 1990 May 4;61(3):447–457. doi: 10.1016/0092-8674(90)90526-k. [DOI] [PubMed] [Google Scholar]
  14. Kuwabara M. D., Sigman D. S. Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry. 1987 Nov 17;26(23):7234–7238. doi: 10.1021/bi00397a006. [DOI] [PubMed] [Google Scholar]
  15. Liu Q., Yong C. B., Astell C. R. In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication. Virology. 1994 Jun;201(2):251–262. doi: 10.1006/viro.1994.1290. [DOI] [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  17. Mueller J. E., Smith D., Bryk M., Belfort M. Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site. EMBO J. 1995 Nov 15;14(22):5724–5735. doi: 10.1002/j.1460-2075.1995.tb00259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Naeger L. K., Cater J., Pintel D. J. The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner. J Virol. 1990 Dec;64(12):6166–6175. doi: 10.1128/jvi.64.12.6166-6175.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Naeger L. K., Salomé N., Pintel D. J. NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells. J Virol. 1993 Feb;67(2):1034–1043. doi: 10.1128/jvi.67.2.1034-1043.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Perros M., Deleu L., Vanacker J. M., Kherrouche Z., Spruyt N., Faisst S., Rommelaere J. Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells. J Virol. 1995 Sep;69(9):5506–5515. doi: 10.1128/jvi.69.9.5506-5515.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roberts M. R., Han Y., Fienberg A., Hunihan L., Ruddle F. H. A DNA-binding activity, TRAC, specific for the TRA element of the transferrin receptor gene copurifies with the Ku autoantigen. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6354–6358. doi: 10.1073/pnas.91.14.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roberts M. R., Miskimins W. K., Ruddle F. H. Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferrin receptor gene and appear to be associated as a heterodimer. Cell Regul. 1989 Nov;1(1):151–164. doi: 10.1091/mbc.1.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sturm R., Baumruker T., Franza B. R., Jr, Herr W. A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer. Genes Dev. 1987 Dec;1(10):1147–1160. doi: 10.1101/gad.1.10.1147. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES