title: 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.
authors.affiliation: Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Lamarche
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D001483:Base Sequence; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C000607892:LIG1 protein, human; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP