Distinct kinetics of human DNA ligases I, IIIα, IIIβ, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

Abstract

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIβ and the hLigIIIα/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/ XRCC4 molecules.

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

“…An unusual feature of purified DNA ligase IV family proteins is that they appear to catalyze only a single ligation event because of inefficient readenylation following ligation (32,42,45). Recently, it has been shown that XLF promotes readenylation of DNA ligase IV-XRCC4, and it was suggested that this readenylation may enable DNA ligase IV-XRCC4 to join both strands at a ligatable DSB (32).…”

“…The DNA ligase IV family is distinct from the other families of eukaryotic DNA ligases in that the purified enzyme is not only predominantly preadenylated but also appears to be capable of completing only a single ligation reaction (32,(42)(43)(44)(45). Because XLF stimulates DNA joining by DNA ligase IV-XRCC4 (31-34, 46), we asked whether Nej1 modulates the catalytic activity of Dnl4-Lif1.…”

Nonhomologous end joining: A good solution for bad ends

“…An already adenylated ligase cannot engage this substrate; this would occur if the “spent” ligase’s active site is re-adenylated in situ , or if the spent ligase is exchanged for another, already adenylated ligase. The former problem might be mitigated somewhat by the general resistance of ligase IV to re-adenylation, relative to other mammalian ligases [127]. Possibly more significantly, aprataxin can remove the adenylate on the substrate regardless of the reason why the ligase is also adenylated; the NHEJ complex is thus reset for another ligation attempt [61, 62].…”

Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor

“…Purified LigI fractions were pooled, concentrated using a 50 kDa MWCO centrifugal filter (EMD Millipore), and then stored at −80°C. LigIIIβ and the LigIV/XRCC4 complex were purified from E. coli and insect cells, respectively as described previously (53,54). …”

“…Nick joining was measured using radioactive- and fluorescence-based assays (53,54). The concentrations of oligonucleotides (Integrated DNA Technologies) DNA were measured using absorbance at 260 nm.…”