title: Identification of a novel motif in DNA ligases exemplified by DNA ligase IV.
abstract: DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, BN9 1RQ UK.
authors.forename: Caterina
authors.initials: C
authors.lastname: Marchetti
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Walker
authors.forename: Federico
authors.initials: F
authors.lastname: Odreman
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vindigni
authors.forename: Aidan J
authors.initials: AJ
authors.lastname: Doherty
authors.forename: Penny
authors.initials: P
authors.lastname: Jeggo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
chemical_list: C000607892:LIG1 protein, human; C000607893:LIG4 protein, human; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP