Home LiteratureArticle Details
PMID: 2191296 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 12 ·1990-06-00 ·Pages 4610-4

Bernad A, Lázaro JM, Salas M, Blanco L

Abstract

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.

MeSH Terms
Amino Acid Sequence Binding Sites Coliphages/enzymology DNA Polymerase II/genetics,metabolism DNA Replication DNA-Directed DNA Polymerase/genetics,metabolism Escherichia coli/enzymology Exodeoxyribonuclease V Exodeoxyribonucleases/metabolism Kinetics Molecular Sequence Data Mutation Sequence Homology, Nucleic Acid
Chemicals
DNA Polymerase II DNA-Directed DNA Polymerase Exodeoxyribonucleases Exodeoxyribonuclease V
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bernad A
Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain.
Lázaro J M
Salas M
Blanco L
References (42)
42 references, click to expand
  1. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins.
    J Mol Biol. 1978 Mar 25;120(1):97-120 PMID: 642007
  2. In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus.
    Mol Gen Genet. 1989 Sep;218(3):523-30 PMID: 2573821
  3. A versatile primer for DNA sequencing in the M13mp2 cloning system.
    Gene. 1980 Jun;10(1):69-73 PMID: 6250947
  4. New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha.
    Cell. 1981 Mar;23(3):647-8 PMID: 6784928
  5. Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.
    Proc Natl Acad Sci U S A. 1982 Sep;79(18):5522-6 PMID: 6813861
  6. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.
    Proc Natl Acad Sci U S A. 1984 Mar;81(6):1639-43 PMID: 6424120
  7. Sequence and structure of yeast phosphoglycerate kinase.
    EMBO J. 1982;1(12):1635-40 PMID: 6765200
  8. Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha.
    Nucleic Acids Res. 1984 Apr 25;12(8):3695-706 PMID: 6728682
  9. Transcription in vitro of phi29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase.
    Eur J Biochem. 1976 Dec;71(1):77-83 PMID: 827446
  10. Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication.
    Proc Natl Acad Sci U S A. 1984 Sep;81(17):5325-9 PMID: 6433348
  11. Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase.
    Proc Natl Acad Sci U S A. 1984 Sep;81(17):5374-8 PMID: 6433349
  12. Bacteriophage phi 29 DNA replication in vitro: participation of the terminal protein and the gene 2 product in elongation.
    Mol Gen Genet. 1984;196(3):381-6 PMID: 6438445
  13. Characterization of a 3'----5' exonuclease activity in the phage phi 29-encoded DNA polymerase.
    Nucleic Acids Res. 1985 Feb 25;13(4):1239-49 PMID: 2987819
  14. Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.
    Nucleic Acids Res. 1985 May 10;13(9):3083-100 PMID: 3158884
  15. A model for the tertiary structure of p21, the product of the ras oncogene.
    Science. 1985 Oct 4;230(4721):78-82 PMID: 3898366
  16. Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides.
    Nucleic Acids Res. 1985 Sep 11;13(17):6331-42 PMID: 3931053
  17. Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.
    Proc Natl Acad Sci U S A. 1985 Oct;82(19):6404-8 PMID: 3863101
  18. Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.
    Nucleic Acids Res. 1985 Nov 11;13(21):7715-28 PMID: 3934646
  19. Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain.
    Proc Natl Acad Sci U S A. 1985 Dec;82(23):7969-73 PMID: 2999787
  20. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.
    EMBO J. 1985 Sep;4(9):2385-8 PMID: 3908095
  21. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.
    Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659-63 PMID: 3012524
  22. Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase.
    Virology. 1986 Sep;153(2):179-87 PMID: 3090778
  23. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene.
    J Virol. 1987 Jan;61(1):125-33 PMID: 3023690
  24. Related functional domains in virus DNA polymerases.
    EMBO J. 1987 Jan;6(1):169-75 PMID: 3034575
  25. Identification by a random sequencing strategy of the fowlpoxvirus DNA polymerase gene, its nucleotide sequence and comparison with other viral DNA polymerases.
    Nucleic Acids Res. 1987 Aug 25;15(16):6563-73 PMID: 2819823
  26. Analysis of sequence-similar pentapeptides in unrelated protein tertiary structures. Strategies for protein folding and a guide for site-directed mutagenesis.
    J Mol Biol. 1987 Sep 20;197(2):331-48 PMID: 3681998
  27. Interference with viral infection by defective RNA replicase.
    J Virol. 1987 Dec;61(12):3946-9 PMID: 3316709
  28. Effect of NH4+ ions on phi 29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase.
    J Virol. 1987 Dec;61(12):3983-91 PMID: 3682063
  29. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.
    EMBO J. 1987 Dec 20;6(13):4219-25 PMID: 3127204
  30. Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I.
    Science. 1988 Apr 8;240(4849):199-201 PMID: 2832946
  31. Structure of concanavalin A at 2.4-A resolution.
    Biochemistry. 1972 Dec 19;11(26):4910-9 PMID: 4638345
  32. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.
    EMBO J. 1988 Jan;7(1):37-47 PMID: 3359994
  33. A new superfamily of replicative proteins.
    Nature. 1988 May 5;333(6168):22-3 PMID: 3362205
  34. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.
    Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772-6 PMID: 3287376
  35. A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination.
    FEBS Lett. 1988 Aug 1;235(1-2):16-24 PMID: 2841153
  36. A sequence motif in many polymerases.
    Nucleic Acids Res. 1988 Nov 11;16(21):9909-16 PMID: 2461550
  37. Cocrystal structure of an editing complex of Klenow fragment with DNA.
    Proc Natl Acad Sci U S A. 1988 Dec;85(23):8924-8 PMID: 3194400
  38. The location, sequence, transcription, and regulation of a baculovirus DNA polymerase gene.
    Virology. 1988 Dec;167(2):591-600 PMID: 3059678
  39. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.
    J Biol Chem. 1989 May 25;264(15):8935-40 PMID: 2498321
  40. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III.
    EMBO J. 1989 Jun;8(6):1849-54 PMID: 2670563
  41. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.
    Cell. 1989 Oct 6;59(1):219-28 PMID: 2790959
  42. Prediction of the secondary structure of proteins from their amino acid sequence.
    Adv Enzymol Relat Areas Mol Biol. 1978;47:45-148 PMID: 364941
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.12.4610
Published
1990-06-00
Pages
4610-4
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC54166
Subset
IM
Grants
NIGMS NIH HHS · 5 R01 GM27242-10 · United States
External Links
PubMed source DOI: 10.1073/pnas.87.12.4610 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2