T4 DNA ligase

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T4 DNA ligase
other names

Polydeoxyribonucleotide synthase [ATP], T4 gene 30 product

Mass / length primary structure 487 amino acids , 55,293 Da
Identifier
External IDs
Enzyme classification
EC, category 6.5.1.1
Orthologue (human)
Entrez 1258680
UniProt P00970
Refseq (protein) NP_049813.1
PubMed search 1258680

The T4 DNA ligase is an enzyme from the group of DNA ligases and is from Enterobakterienphagen-T4 formed.

properties

The two DNA double strands are covalently linked by the ligase

The T4 DNA ligase is produced in infected E. coli at the beginning of the lytic cycle of the T4 phage. It creates a phosphodiester bond between two adjacent DNA sequences in DNA double strands and is used by the bacteriophages for DNA replication , recombination and DNA repair . Cofactors are divalent magnesium ions . The T4 DNA ligase combines both blunt ends and sticky ends of double-stranded DNA , RNA and DNA-RNA hybrids, but only to a small extent single-stranded DNA or RNA. The double strand must have at least 5 paired nucleobases . A T4 DNA ligase modified by protein design , which was produced as a fusion protein with NFκB p50 , has a 160-fold increased enzyme activity .

The T4 DNA ligase catalyzes the reaction:

ATP + (deoxyribonucleotide) (n) -3'-hydroxyl + 5'-phospho- (deoxyribonucleotide) (m) (deoxyribonucleotide) (n + m) + AMP + diphosphate

Applications

The T4 DNA ligase is as part of a cloning or an artificial gene synthesis for the ligation of two DNA duplexes to each other (with 1 mM ATP and 10 mM Mg 2+ ). Sticky ends are ligated around twelve times faster than blunt ends (10 min vs. 2 h). A thermostable DNA ligase for use in polymerase chain reaction- based methods is the Taq DNA ligase from Thermus aquaticus .

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, table of contents, PMID 12626685 , PMC 150520 (free full text).

Web links

Individual evidence

  1. ↑ J. Armstrong, RS Brown, A. Tsugita: Primary structure and genetic organization of phage T4 DNA ligase. In: Nucleic acids research. Volume 11, Number 20, October 1983, pp. 7145-7156, PMID 6314278 , PMC 326445 (free full text).
  2. ^ H. Kuhn, MD Frank-Kamenetskii: Template-independent ligation of single-stranded DNA by T4 DNA ligase. In: The FEBS journal. Volume 272, Number 23, December 2005, pp. 5991-6000, doi : 10.1111 / j.1742-4658.2005.04954.x , PMID 16302964 .
  3. ↑ DR Horspool, RJ Coope, RA Holt: Efficient assembly of very short oligonucleotides using T4 DNA ligase. In: BMC research notes. Volume 3, November 2010, p. 291, doi : 10.1186 / 1756-0500-3-291 , PMID 21062485 , PMC 2994885 (free full text).
  4. ↑ RH Wilson, SK Morton, H. Deiderick, ML Gerth, HA Paul, I. Gerber, A. Patel, AD Ellington, SP Hunicke-Smith, WM Patrick: Engineered DNA ligases with improved activities in vitro. In: Protein engineering, design & selection: PEDS. Volume 26, Number 7, July 2013, pp. 471-478, doi : 10.1093 / protein / gzt024 , PMID 23754529 .
  5. ↑ IR Lehman: DNA ligase: structure, mechanism, and function. In: Science. Volume 186, Number 4166, November 1974, pp. 790-797, PMID 4377758 .