Abstract
Full-length cDNAs for DNA ligase IV and the alpha and beta isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase III alpha and III beta share a common NH(2) terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the NH(2) terminus of DNA ligase IV does not. Reverse transcriptase-polymerase chain reaction analyses with adult frog tissues demonstrate that while DNA ligase III alpha and DNA ligase IV are ubiquitously expressed, DNA ligase III beta expression is restricted to testis and ovary. Mitochondrial lysates from X. laevis oocytes contain both DNA ligase III alpha and III beta but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis, and in vitro cross-linking experiments demonstrate that mtDNA ligase III alpha exists as a high molecular weight complex. We discuss the possibility that DNA ligase III alpha exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.
Highlights
Most eukaryotic cells rely on mitochondria for many vital cellular processes such as the generation of adenosine triphosphate (ATP), the regulation of the cytoplasmic redox state, heat production, apoptosis, and the synthesis of amino acids, pyrimidines, heme, aldosterone, cortisol, sex steroids, and many other key metabolites
The predicted translation products beginning with the first AUG codons encountered in the frog cDNAs contain potential amino-terminal mitochondrial localization signals, as described for human DNA ligase III cDNAs in the Introduction
This paper provides the sequences of both Xenopus DNA ligase III and IV along with more definitive immunological evidence for the mitochondrial localization of DNA ligase III
Summary
Most eukaryotic cells rely on mitochondria for many vital cellular processes such as the generation of adenosine triphosphate (ATP), the regulation of the cytoplasmic redox state, heat production, apoptosis, and the synthesis of amino acids, pyrimidines, heme, aldosterone, cortisol, sex steroids, and many other key metabolites. The mtDNA ligase activity purified from Xenopus oocyte mitochondria has a size and substrate specificity similar to those of human DNA ligase III or IV. In Vitro Cross-linking—One-microliter samples of partially purified DNA ligase III␣ and III activities from X. laevis oocyte mitochondria and human nuclear DNA ligase III were adenylated as described above.
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