DNA LIGASE FROM EMBRYOS OF DROSOPHILA MELANOGASTER (NUCLEIC ACIDS)
RABIN, BRUCE ARLAN
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DNA ligase is an enzyme that acts during DNA replication, recombination, and repair to seal single-stranded interruptions in the backbone of duplex DNA. To more fully understand how this enzyme functions in a highly differentiated eukaryotic organism during these complex processes, DNA ligase has been isolated from 6-12 hour embryos of Drosophila melanogaster and its properties have been analyzed. There is at least 5-10 fold more ligase activity present during embryogenesis than at any other time in development, presumably because the extremely high rate of DNA replication in cleavage-stage nuclei (18,000 kilobases/minute('-1) molecule('-1)) requires enormous amounts of DNA metabolic enzymes.;The purified protein (>97% homogeneous) has a native molecular weight of 79,800 and was a monomer in solution. The enzyme is ATP-dependent (the apparent K(,m) is 1.6 (mu)M) and is competitively inhibited by dATP (the apparent K(,i) is 2.3 (mu)M). The Drosophila enzyme acts by catalyzing the formation of 3', 5'-phosphodiester bonds at nicks in duplex DNA. By isolating reaction intermediates and characterizing partial reactions, the overall joining reaction was shown to proceed in 3 discreet steps. First, the enzyme forms a covalent ligase-adenylylate complex in the absence of DNA. The adenylyl group is then transferred from the enzyme to the 5'-phosphoryl terminus of the nick, forming a high energy 5', 5'-pyrophosphate bond. In the final step, the 3'-hydroxyl terminus of the nick attacks the 5'-phosphoryl group and a phosphodiester bond is formed, releasing free AMP. The enzyme can also catalyze the reverse reaction and thus, in the presence of high concentrations of enzyme and AMP, acts like an AMP-dependent nicking-closing enzyme.;In addition to joining DNA (the apparent K(,m) for termini of p(dT)(,20)(.)poly(dA) is 1.0 (mu)M), the Drosophila DNA ligase catalyzes a broad range of other polynucleotide joining reactions. These include the joining of RNA to RNA, DNA to RNA in either orientation, and DNA containing a single nucleotide mismatch at the 3'-terminus, when these substrates are aligned on a DNA template. The Drosophila enzyme also joins DNA with short, protruding termini, and in the presence of PEG, this enzyme can efficiently join fully base-paired (blunt-ended) DNA. No joining was detected with any substrate when RNA was used as the template.
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