title: Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase.
abstract: Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Poulami
authors.initials: P
authors.lastname: Samai
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053904:DNA Breaks, Single-Stranded; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D000249:Adenosine Monophosphate; D004247:DNA; D009713:Nucleotidyltransferases; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases