T4 DNA polymerase

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DNA-directed DNA polymerase
DNA-directed DNA polymerase
Ribbon model of T4 DNA polymerase according to PDB  1NOY
other names

Gene product 43, Gp43

Existing structure data : PDB  1NOY , PDB  1NOZ

Mass / length primary structure 898 amino acids , 103,610 Da
Identifier
External IDs
Enzyme classification
EC, category 2.7.7.7
Orthologue (bacteriophage T4)
Entrez 1258685
UniProt P04415
Refseq (protein) NP_049662.1
PubMed search 1258685

T4 DNA polymerase is an enzyme from the group of DNA polymerases that is encoded by gene 43 of bacteriophage T4 .

properties

The T4 DNA polymerase is used by the bacteriophage T4 to replicate the viral genome . T4 DNA polymerase has two enzyme activities: a polymerase activity, which catalyzes DNA synthesis from the 5 'to the 3' end , and a 3 '→ 5' exonuclease activity, the so-called proofreading function. proof reading ), which makes it possible to recognize the incorporation of an unsuitable nucleotide and then to remove it again from the DNA. In contrast to DNA polymerase I from E. coli , it lacks 5 '→ 3' exonuclease activity. The T4 DNA polymerase always uses an existing single strand of DNA as a template (template) for the synthesis of a new, complementary strand. A 5 'DNA overhang and a high concentration of dNTPs are required for polymerase activity . High concentrations of dNTPs inhibit exonuclease activity. The exonuclease activity shows an approximately 100-fold increased preference for single strands compared to double-stranded DNA. Divalent magnesium ions are used as a cofactor .

function

The bacteriophage T4 codes for all proteins that it needs for its replication . In the 1960s, it was possible to create gene maps for the T4 genome . In 1964, the identification of gene 43 as the structural gene for T4 DNA polymerase, which is therefore also called T4 gp43 DNA polymerase. T4 DNA polymerase is one of ten proteins of the T4 replisome , which also includes T4 DNA ligase . T4 DNA polymerase, like the eukaryotic Pol α, belongs to the Pol B family. Gp45 from bacteriophage T4 is used as a DNA clamp .

Applications

The T4 DNA polymerase is used in molecular biology to remove 3 'overhangs and for filling (fill-in) of 5' overhangs to generate blunt ends ( blunt ends ) used. This function is used when cloning DNA molecules that have been generated with restriction enzymes . The enzyme is also used to label DNA molecules, using the combination of exonuclease and polymerase activity. One advantage of T4 DNA polymerase in second strand synthesis during cDNA cloning is its high accuracy. It is used in site-specific DNA mutagenesis because of the lack of strand displacement activity.

literature

  • ES Miller, E. Kutter, G. Mosig, F. Arisaka, T. Kunisawa, W. Rüger: Bacteriophage T4 genome. In: Microbiology and molecular biology reviews: MMBR. Volume 67, Number 1, March 2003, pp. 86-156, PMID 12626685 , PMC 150520 (free full text).

Web links

Individual evidence

  1. ↑ DNA Polymerase, T4 - Worthington Enzyme Manual. In: worthington-biochem.com. Retrieved December 10, 2016 .
  2. ↑ WM Huang, IR Lehman: On the exonuclease activity of phage T4 deoxyribonucleic acid polymerase. In: The Journal of Biological Chemistry. Volume 247, Number 10, May 1972, pp. 3139-3146, PMID 4554914 .
  3. ↑ RS EDGAR, I. LIELAUSIS: TEMPERATURE-SENSITIVE MUTANTS OF BACTERIOPHAGE T4D: THEIR ISOLATION AND GENETIC CHARACTERIZATION. In: Genetics. Volume 49, April 1964, pp. 649-662, PMID 14156925 , PMC 1210603 (free full text).
  4. ↑ RS EDGAR, GH DENHARDT, RH EPSTEIN: A COMPARATIVE GENETIC STUDY OF CONDITIONAL LETHAL MUTATIONS OF BACTERIOPHAGE T4D. In: Genetics. Volume 49, April 1964, pp. 635-648, PMID 14156924 , PMC 1210602 (free full text).
  5. ↑ A. De Waard, AV Paul, IR Lehman: The structural gene for deoxyribonucleic acid polymerase in bacteriophages T4 and T5. In: Proc. Natl. Acad. Sci. USA Volume 54, Number 4, October 1965, pp. 1241-1248, PMID 5219829 , PMC 219850 (free full text).
  6. ↑ TC Mueser, JM Hinerman, JM Devos, RA Boyer, KJ Williams: Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives. In: Virol. J. Volume 7, December 2010, p. 359, doi : 10.1186 / 1743-422X-7-359 , PMID 21129204 , PMC 3012046 (free full text) (review).
  7. ↑ JD Karam, WH Konigsberg: DNA polymerase of the T4-related bacteriophages. In: Prog. Nucleic Acid Res. Mol. Biol. Vol. 64, 2000, pp. 65-96, PMID 10697407 (review).
  8. ↑ KC Deen, TA Landers, M. Berninger: Use of T4 DNA polymerase replacement synthesis for specific labeling of plasmid-cloned inserts. In: Anal. Biochem. Volume 135, Number 2, December 1983, pp. 456-465, PMID 6318604 .