DNA ligase from Drosophila melanogaster embryos. Substrate specificity and mechanism of action.

Bruce Rabin, J. W. Chase

Research output: Contribution to journalArticle

Abstract

DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B. A., Hawley, R. S., and Chase, J. W. (1986) J. Biol. Chem. 261, 10637-10645). This enzyme had an apparent Km for ATP of 1.6 microM. Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction. The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM. The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM. Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA. In addition, p(dT)7U could be joined when annealed with poly(dA). No joining was detected when RNA served as the template. Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA. The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases. The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible. Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.

Original languageEnglish (US)
Pages (from-to)14105-14111
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number29
StatePublished - Oct 15 1987
Externally publishedYes

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DNA Ligases
Substrate Specificity
Drosophila melanogaster
Embryonic Structures
Joining
DNA
Substrates
RNA
Enzymes
Hydroxyl Radical
Drosophila
Nucleotides
Adenosine Triphosphate
Circular DNA
Molecules
Adenosine Monophosphate
Homopolymerization
Oligonucleotides
Adenosine

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA ligase from Drosophila melanogaster embryos. Substrate specificity and mechanism of action. / Rabin, Bruce; Chase, J. W.

In: Journal of Biological Chemistry, Vol. 262, No. 29, 15.10.1987, p. 14105-14111.

Research output: Contribution to journalArticle

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