[Ala107]-MBP (104-118) The absence of the LIG gene
The absence of the LIG3 gene in yeasts has prevented the use of genetically tractable lower eukaryotes, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, as models to gain insights into the cellular functions of and interplay between the DNA ligases encoded by the LIG1, LIG3 and LIG4 genes in higher eukaryotes. Based on the efforts of many laboratories, there is an emerging picture of functional redundancy among the DNA ligases in mammalian [Ala107]-MBP (104-118) that is complicated by the LIG3 gene encoding multiple DNA ligase polypeptides. In this review, we focus on the structure and function of the DNA ligases encoded by the mammalian LIG3 gene.
The human LIG3 gene is located on human chromosome 17 at q11.2–q12 (Chen et al., 1995, Wei et al., 1995). Unlike the LIG1 and LIG4 genes, the LIG3 gene encodes three or possibly four different DNA ligase polypeptides (Fig. 1). Mitochondrial and nuclear versions of DNA ligase IIIα are generated in all cells by alternative translation initiation (Lakshmipathy and Campbell, 1999). The DNA ligase IIIα mRNA open reading frame encodes an N-terminal mitochondrial leader sequence (MLS) that is cleaved off during entry into mitochondria (Fig. 2). Thus, translation initiation at the first ATG of the full-length open reading frame generates mitochondrial DNA ligase IIIα whereas translation initiation at an internal ATG adjacent to a Kozak consensus sequence generates nuclear DNA ligase IIIα (Chen et al., 1995, Lakshmipathy and Campbell, 1999, Wei et al., 1995). Since there is no obvious NLS (NLS) within the DNA ligase IIIα polypeptide, it has been suggested, as shown in Fig. 2, that nuclear localization is dependent upon complex formation with a partner protein XRCC1 that does have a nuclear localization signal (Caldecott, 2003, Parsons et al., 2010). The interaction of DNA ligase IIIα with XRCC1 and other partner proteins is described below. Prior to the cloning of the human LIG genes, biochemical studies had identified a 70kDa DNA ligase in addition to a 125kDa DNA ligase I and a 100kDa DNA ligase III that was designated DNA ligase II (Soderhall and Lindahl, 1975, Tomkinson et al., 1991). Amino acid sequencing of peptides from purified DNA ligase II revealed that this polypeptide was encoded by the LIG3 gene and is most likely generated by proteolysis of DNA ligase IIIα during purification (Chen et al., 1995, Husain et al., 1995, Wang et al., 1994). Thus, the confusing nomenclature of the mammalian DNA ligases can be attributed to a purification artifact during attempts to purify and characterize these enzymes. The LIG3 gene is ubiquitously expressed at low levels in all human tissues and cells except for the testes where expression levels are about 10-fold higher (Chen et al., 1995, Wei et al., 1995). Further analysis revealed that the elevated levels of expression occur in primary spermatocytes undergoing recombination prior to the first meiotic division and that a distinct mRNA species, DNA ligase IIIβ, is generated by an alternative splicing mechanism that has only been detected in male germ cells (Mackey et al., 1997). The alternative splicing, which replaces the exon encoding the C-terminal 77 amino acids of DNA ligase IIIα with a novel 17- to 18-amino acid sequence (Fig. 1), begins in early pachytene spermatocytes with DNA ligase IIIβ mRNA detectable throughout pachytene and in round spermatids (Mackey et al., 1997). Since the 5′ end of DNA ligase IIIβ mRNA is identical to that of DNA ligase IIIα mRNA, this transcript is also capable of encoding nuclear and mitochondrial versions of DNA ligase IIIβ by alternative translation (Fig. 1). It should be noted that DNA ligase IIIβ mRNA has not been detected in the ovary (Mackey et al., 1997). This may reflect differences between oogenesis and spermatogenesis. Furthermore, organisms such as S. cerevisiae, that lack a LIG3 homolog undergo meiosis. Thus, although the expression pattern of DNA ligase IIIβ during spermatogenesis suggests that this enzyme participates in the completion of homologous recombination events that occur during meiotic prophase, this has not yet been definitively demonstrated. Conceivably, DNA ligase IIIβ may be involved in maintaining genomic integrity when histones are replaced with protamines late in the haploid phase of spermatogenesis.