DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.
"dependent DNA ligases demonstrate two distinct domains: the adenylation domain and the oligonucleotide-binding (OB)-fold domain (Kim et al. 2009; Akey et al. 2006; Pascal et al. 2004; Odell et al. 2000; Subramanya et al. 1996). According to these structures, the catalytic core domains adopt an open form, which locates motif VI at a distant position from the AMP-binding site; an essential step for ATP hydrolysis. "
[Show abstract][Hide abstract] ABSTRACT: DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.
World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology) 06/2013; 29(12). DOI:10.1007/s11274-013-1386-1 · 1.78 Impact Factor
"Although putative homologues of Ku as well as ATP-dependent DNA ligases have been reported in bacteria, bacterial NHEJ also apparently proceeds in the absence of XRCC4, XLF, and DNA-PKcs (Pitcher et al. 2007; Shuman and Glickman 2007). Yet, as shown by results in mycobacteria that have a robust NHEJ repair pathway requiring Ku but with ligase fused to polymerase, these pathogens have a shocking 50% error rate for NHEJ (Akey et al. 2006; Aniukwu et al. 2008; Gong et al. 2005) that may reflect the importance of the extra eukaryotic components for fidelity. As a general theme, filaments provide geometric control of DNA pieces, as seen for bacterial pili that promote bacterial uptake of DNA for transformation (Hartung et al. 2011) . "
[Show abstract][Hide abstract] ABSTRACT: DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.
"Thus, the selectivity of the LigD POL for manganese (or cobalt) over magnesium is not explained by reliance on soft amino acid metal ligands. The structure of the LigD LIG domain suggests that carboxylates are the likely metal ligands (21), as they are for all other ligases that utilize magnesium as the preferred cofactor. At this point, it is not clear why bacterial NHEJ ligases prefer manganese over magnesium, though we suspect it may relate to their distinctive preference for sealing broken nucleic acids with 3′-monoribonucleotide ends (13). "
[Show abstract][Hide abstract] ABSTRACT: LigD 3'-phosphoesterase (PE) enzymes perform end-healing reactions at DNA breaks. Here we characterize the 3'-ribonucleoside-resecting activity of Candidatus Korarchaeum PE. CkoPE prefers a single-stranded substrate versus a primer-template. Activity is abolished by vanadate (10 mM), but is less sensitive to phosphate (IC(50) 50 mM) or chloride (IC(50) 150 mM). The metal requirement is satisfied by manganese, cobalt, copper or cadmium, but not magnesium, calcium, nickel or zinc. Insights to CkoPE metal specificity were gained by solving new 1.5 Å crystal structures of CkoPE in complexes with Co(2+) and Zn(2+). His9, His15 and Asp17 coordinate cobalt in an octahedral complex that includes a phosphate anion, which is in turn coordinated by Arg19 and His51. The cobalt and phosphate positions and the atomic contacts in the active site are virtually identical to those in the CkoPE·Mn(2+) structure. By contrast, Zn(2+) binds in the active site in a tetrahedral complex, wherein the position, orientation and atomic contacts of the phosphate are shifted and its interaction with His51 is lost. We conclude that: (i) PE selectively binds to 'soft' metals in either productive or non-productive modes and (ii) PE catalysis depends acutely on proper metal and scissile phosphate geometry.
Nucleic Acids Research 09/2011; 40(2):828-36. DOI:10.1093/nar/gkr767 · 9.11 Impact Factor
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