Human DNA Ligase IV and the Ligase IV/XRCC4 Complex:  Analysis of Nick Ligation Fidelity

Abstract

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/ intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in Vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.In addition to sealing broken DNA back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of the repair pathways they are involved in. This is well illustrated by the example of mammalian base excision repair (BER 1 ), where nicks containing 3′-OH mismatched base pairs, resulting from aberrant gap-filling by DNA polymerase (Pol ), are sealed inefficiently by the DNA ligase III/XRCC1 complex (1). This delay in mismatch ligation is expected to provide a greater window of opportunity for nick editing by the 3′ f 5′ exonuclease activity of APE1 (1, 2), the predominant mammalian AP endonuclease (APE).Though a large body of qualitative and quantitative data suggests that DNA ligases from a broad spectrum of organisms are intolerant of non-Watson-Crick base pairing schemes at the 3′-OH terminus of a nick (3 and references therein), our recent analysis of the African swine fever virus (ASFV)-encoded DNA ligase demonstrated that analogous to the recently discovered error-prone DNA polymerases, there are exceptions to this rule (3). The ASFV DNA ligase is not only tolerant of numerous 3′ mismatched base pairing schemes but also actually displays higher catalytic efficiency for sealing a 3′ C:T 2 mismatch than it does for sealing nicks containing Watson-Crick base pairs. Whether the errortolerance of t...

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.…”

Computational analysis of DNA repair pathways in breast cancer

“…Besides, to obtain a gene-level measurement, RNA-seq usually adopts the union of the transcripts that represent the same gene while microarray often considers only the mostabundant probe of the gene. However, recent large-scale comparisons showed that these two techniques produce highly-correlated results (Spearman correlation coefficient of 0.8) (88) or that the choice between them is not a significant factor affecting the final results (89). Together with the facts that microarray is more cost-effective, and that a great wealth of microarray-based expression data has already been accumulated, the results from these two gene expression profiling techniques are complementary to each other.…”

“…PIN1 controls CtIP levels by promoting its isomerization in a CDK2-dependent manner followed by poly-ubiquitination (through an as-yet-unknown E3 ubiquitin ligase) and consequent degradation to limit end resection (88). Although PCNA (proliferating cell nuclear antigen) has recently been proposed to facilitate long-range resection by promoting the function of EXO1 (93), in general the regulatory mechanisms involved in this step are not well-understood.…”

“…In this complex, XRCC4 seems to stabilize LIGIV and stimulates its joining activity [87,88]; XLF may help LIGIV to ligate a more diverse array of DNA [89]. This complex is targeted to damage sites by an interaction with Ku [90,91].…”