br The related catalytic regions of the human
The related catalytic regions of the human DNA ligases contain three domains, a DNA binding domain (DBD), a nucleotidyl transferase domain (NTase) and an oligonucleotide/oligosaccharide-fold binding domain (OBD) (Ellenberger and Tomkinson, 2008). Similar to DNA ligase I, the DNA ligase III polypeptide adopts a flexible, extended conformation in the absence of DNA (Cotner-Gohara et al., 2010). When it engages a DNA nick, the domains of the catalytic region contact and encircle the nicked DNA in a compact, closed clamp structure shown in Fig. 3A (Cotner-Gohara et al., 2010). The protein architecture and conformation of the catalytic domains in this structure are remarkably similar to those of the DNA ligase I catalytic domains bound to nicked DNA despite these polypeptides only sharing 21% amino tpca identity (Cotner-Gohara et al., 2010). A unique feature of the DNA ligases encoded by the LIG3 gene compared with the other human DNA ligases is an N-terminal zinc finger (ZnF) (Fig. 1). This ZnF is structurally related to the pair of ZnFs at the N-terminus of poly(ADP-ribose) polymerase 1 (PARP1) that facilitate binding of PARP1 to DNA breaks and other abnormal DNA structures (Mackey et al., 1999). Although the DNA ligase III ZnF also binds to DNA breaks, it is not required for nick ligation but does enable the enzyme to efficiently join nicked DNA at high salt concentrations (Mackey et al., 1999). Interestingly, the DNA ligase III ZnF and DBD cooperate to form a nick-binding module with the NTase and OBD forming a second nick-binding module (Cotner-Gohara et al., 2008). These two modules have different DNA binding properties with ZnF/DBD module being more tolerant of different nick structures, including gaps, whereas the NTase/OBD module preferentially binds to ligatable nicks (Cotner-Gohara et al., 2008). Based on these properties and biophysical studies, a jackknife model for the ligation of DNA nicks by DNA ligase III has been proposed (Cotner-Gohara et al., 2008, Cotner-Gohara et al., 2010). In this model, the ZnF/DBD module acts as a nick sensor that initially engages DNA breaks with the DNA ligase III polypeptide in an extended conformation (Fig. 3B). If the nick is ligatable, the DNA ligase III polypeptide undergoes a conformational change with NTase/OBD displacing the ZnF/DBD module and then forming the compact, closed clamp structure with the DBD around the DNA nick (Fig. 3B). Among the human DNA ligases, DNA ligase III has the most robust intermolecular DNA joining activity (Chen et al., 2000). This activity is not only dependent upon the ZnF but also involves key residues within the DBD (Cotner-Gohara et al., 2008, Cotner-Gohara et al., 2010). It has been proposed that the NTase/OBD and ZnF/DBD modules each engage a DNA end (Cotner-Gohara et al., 2010), enabling DNA ligase III to juxtapose and ligate the DNA ends. Alternatively, it is possible that intermolecular ligation involves an interaction in trans between the ZnF and DBD of two DNA ligase III molecules, each bound to a DNA end. At the C-terminus of DNA ligase IIIα is a breast cancer susceptibility protein (BRCA) 1-related C-terminal (BRCT) domain (Bork et al., 1997). This domain, which is about 100 amino acids long, has been found in many proteins involved in DNA repair and the DNA damage response, and is often involved in protein:protein interactions (Bork et al., 1997). In the case of nuclear DNA ligase IIIα, the BRCT domain interacts with the DNA repair protein, X-ray repair cross-complementing protein 1 (XRCC1) (Fig. 2A), an interaction that is described in more detail below. As a result of the alternative splicing event, the majority of the BRCT domain is missing from DNA ligase IIIβ, which does not interact with XRCC1. The short additional amino acid sequence at the C-terminus of DNA ligase IIIβ acts as a NLS (Fig. 1).
Protein partners of DNA ligase III
Following the identification of the interaction between DNA ligase IIIα and XRCC1 (Caldecott et al., 1994), it was assumed that DNA ligase IIIα participated in base excision repair and the repair of SSBs with XRCC1 and that mutational inactivation of the LIG3 gene would result in the same phenotype as xrcc1 mutant cells. However, expression of a mutant version of XRCC1, which did not interact with DNA ligase IIIα and, as a consequence, did not increase the steady state levels of nuclear DNA ligase IIIα, complemented the DNA damage sensitivity of cycling xrcc1 mutant cells (Taylor et al., 2000a). These studies demonstrated that XRCC1, which is present at higher levels than DNA ligase IIIα (Leppard et al., 2003), functions independently of DNA ligase IIIα in nuclear DNA repair. Furthermore, studies by the Campbell laboratory showed that, although XRCC1 was not detectable in mitochondria, the reduction of DNA ligase IIIα levels by siRNA disrupted mitochondrial function, indicating that the mitochondrial version of DNA ligase IIIα functions in mitochondrial DNA metabolism independently of XRCC1 (Lakshmipathy and Campbell, 2000, Lakshmipathy and Campbell, 2001).