Home LiteratureArticle Details
PMID: 10339555 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

Comparison of the 5' nuclease activities of taq DNA polymerase and its isolated nuclease domain.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 96 ·No. 11 ·1999-05-25 ·Pages 6143-8

Lyamichev V, Brow MA, Varvel VE, Dahlberg JE

Abstract

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5' nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5' arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5' nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3' arm of the bifurcation; optimal stimulation requires overlap of the 3' terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.

MeSH Terms
Base Sequence DNA/chemistry,metabolism Endodeoxyribonucleases/metabolism Exodeoxyribonuclease V Exodeoxyribonucleases/chemistry,metabolism Mutagenesis, Site-Directed Nucleic Acid Conformation Peptide Fragments/metabolism Recombinant Proteins/chemistry,metabolism Sequence Deletion Substrate Specificity Taq Polymerase/chemistry,metabolism Thermus/enzymology
Chemicals
Peptide Fragments Recombinant Proteins DNA Taq Polymerase Endodeoxyribonucleases Exodeoxyribonucleases Exodeoxyribonuclease V
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Lyamichev V
Department of Biomolecular Chemistry, 1300 University Avenue, University of Wisconsin, Madison, WI 53706, USA.
Brow M A
Varvel V E
Dahlberg J E
References (36)
36 references, click to expand
  1. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.
    Mol Cell Biol. 1994 Jul;14(7):4878-88 PMID: 8007985
  2. The characterization of a mammalian DNA structure-specific endonuclease.
    EMBO J. 1994 Mar 1;13(5):1235-46 PMID: 8131753
  3. XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair.
    Nature. 1994 Sep 29;371(6496):432-5 PMID: 8090225
  4. Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.
    J Immunol Methods. 1995 Jun 14;183(1):15-26 PMID: 7602133
  5. Crystal structure of Thermus aquaticus DNA polymerase.
    Nature. 1995 Aug 17;376(6541):612-6 PMID: 7637814
  6. Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage.
    J Biol Chem. 1995 Dec 22;270(51):30377-83 PMID: 8530463
  7. Enzymatic synthesis of deoxyribonucleic acid. XXIX. Hydrolysis of deoxyribonucleic acid from the 5' terminus by an exonuclease function of deoxyribonucleic acid polymerase.
    J Biol Chem. 1969 Jun 10;244(11):3029-37 PMID: 4890763
  8. Deoxyribonuclease IV: a new exonuclease from mammalian tissues.
    Proc Natl Acad Sci U S A. 1969 Feb;62(2):597-603 PMID: 5256235
  9. The conformation of chicken, rat and human U1A RNAs in solution.
    Nucleic Acids Res. 1981 Feb 25;9(4):841-58 PMID: 6164982
  10. Transient generation of displaced single-stranded DNA during nick translation.
    Cell. 1982 Nov;31(1):53-60 PMID: 6218882
  11. Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli.
    Gene. 1987;51(2-3):255-67 PMID: 3110013
  12. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.
    J Biol Chem. 1989 Apr 15;264(11):6427-37 PMID: 2649500
  13. Calculation of protein extinction coefficients from amino acid sequence data.
    Anal Biochem. 1989 Nov 1;182(2):319-26 PMID: 2610349
  14. Use of T7 RNA polymerase to direct expression of cloned genes.
    Methods Enzymol. 1990;185:60-89 PMID: 2199796
  15. Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli.
    J Biol Chem. 1990 Aug 25;265(24):14579-91 PMID: 2201688
  16. Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.
    Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2724-8 PMID: 8610108
  17. Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins.
    Cell. 1996 Jun 28;85(7):1101-12 PMID: 8674116
  18. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease.
    Nature. 1996 Jul 4;382(6586):90-3 PMID: 8657312
  19. In vivo selection of RNAs that localize in the nucleus.
    EMBO J. 1997 Feb 17;16(4):793-806 PMID: 9049308
  20. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair.
    Bioessays. 1997 Mar;19(3):233-40 PMID: 9080773
  21. The crystal structure of flap endonuclease-1 from Methanococcus jannaschii.
    Nat Struct Biol. 1998 Aug;5(8):707-13 PMID: 9699635
  22. Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.
    Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12562-7 PMID: 9770525
  23. Structure-specific DNA cleavage by 5' nucleases.
    Trends Biochem Sci. 1998 Sep;23(9):331-6 PMID: 9787638
  24. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.
    Nat Biotechnol. 1999 Mar;17(3):292-6 PMID: 10096299
  25. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.
    Cell. 1998 Oct 2;95(1):135-46 PMID: 9778254
  26. Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase.
    Nucleic Acids Res. 1990 Dec 25;18(24):7317-22 PMID: 2175431
  27. Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.
    J Biol Chem. 1991 Jan 25;266(3):1888-97 PMID: 1703156
  28. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli.
    Anal Biochem. 1990 Dec;191(2):396-400 PMID: 2085185
  29. A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease.
    Nucleic Acids Res. 1991 Aug 11;19(15):4127-32 PMID: 1651477
  30. A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.
    Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9377-81 PMID: 1329095
  31. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.
    Nucleic Acids Res. 1993 Mar 25;21(6):1345-9 PMID: 8464724
  32. Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.
    Nature. 1993 May 13;363(6425):182-5 PMID: 8483504
  33. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases.
    Science. 1993 May 7;260(5109):778-83 PMID: 7683443
  34. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.
    Mol Cell Biol. 1993 Oct;13(10):6393-402 PMID: 8413238
  35. Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease.
    Nature. 1993 Nov 25;366(6453):365-8 PMID: 8247134
  36. Monoclonal antibodies prepared against the DNA polymerase from Thermus aquaticus are potent inhibitors of enzyme activity.
    J Immunol Methods. 1994 Jun 24;172(2):147-63 PMID: 7518481
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.96.11.6143
Published
1999-05-25
Pages
6143-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC26849
Subset
IM
Grants
NIGMS NIH HHS · T32 GM008349 · United States
NIGMS NIH HHS · GM08349 · United States
NIGMS NIH HHS · GM30230 · United States
External Links
PubMed source DOI: 10.1073/pnas.96.11.6143 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