The Human DNA Ligase III Gene Encodes Nuclear and Mitochondrial Proteins

Abstract

We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.For some time it was thought that the mammalian mitochondria lacked the capacity to repair damaged DNA (18). This hypothesis had its origin in the observation that pyrimidine dimers produced within the mitochondrial DNA (mtDNA) of cultured cells were not repaired, in marked contrast to similar lesions produced in the nuclear genomes of these same cells (4,15,21). However, it is now clear that this organelle is not entirely deficient in DNA repair activity. For example, it appears that base excision repair of oxidized DNA occurs in mtDNA of mammalian cells (8,23,34,40). In addition, a number of studies have revealed that mammalian cultured cells can repair mtDNA damage caused by a number of chemical agents, including cisplatin (15), bleomycin (33), streptozotocin (22), and 4-nitroquinoline (36). More recently, it was shown that mammalian mitochondrial extracts possess DNA nonhomologous end-joining and homologous recombination activities (14, 41). Taken together, these findings indicate that mammalian mitochondria likely possess a number of DNA repair pathways. A series of recent observations suggest that mutations to the mitochondrial genome may be associated with a variety of human pathologies (5, 9, 10, 38, 49), thus highlighting the need for a greater understanding of the molecular...

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria

“…Ligase III immunodepletion diminished the production of the final 36-mer repair product (Fig. 3C, lane 7), consistent with the Ligase III activity in mitochondria (12,54). The small residual 36-mer ligated product in Fig.…”

“…Moreover, the mitochondrial genome does not encode DNA repair proteins, and is thus completely dependent on proteins encoded in the nucleus for its repair and integrity. The corresponding genes encode proteins for both nuclear and mitochondrial repair (12)(13)(14). Yet, the full spectrum of nuclear DNA repair proteins involved in mtDNA repair is not entirely known.…”

Dual localization of human DNA topoisomerase IIIα to mitochondria and nucleus

“…The significance of these observations is not clear, and it is plausible that nuclear transport of proteins might be defective in transfected cells that exhibited exclusive mitochondrial localization of hTop3␣(1-1001)-EGFP. This type of uneven partition of a protein in the mitochondria and nuclei of different populations of transfected cells was observed for human DNA ligase III (34). information alone could not answer the question whether a significant fraction of endogenous DNA topoisomerase III␣ would be localized to the mitochondria.…”

Bovine mtDNA D-loop haplotypes exceed mutations in number despite reduced recombination: an effective alternative for identity control

“…The mitochondrial nature of this latter enzyme is evident from its first 87 amino acids, which are analogous to the double-methionine structure (86 residues, 65% identity and 77% similarity) at the amino terminus of human LIG3. The second methionine has been designated as the first residue for the nuclear LIG3 protein, while the first one is followed by a putative mitochondrialtargeting sequence and is exploited for the mitochondrial isoform (Lakshmipathy and Campbell, 1999). Another line of evidence for a possible effect of mtDNA recombination is the demonstrated decline in linkage disequilibrium as a function of distance between polymorphic sites in some ruminant populations (Slate and Phua, 2003).…”