Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase Journal Article
| Authors: | Samai, P.; Shuman, S. |
| Article Title: | Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase |
| Abstract: | Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structureguided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; nonhuman; dna polymerase; protein domain; amino acid substitution; serine; in vivo study; in vitro study; enzyme activity; structure activity relation; mutational analysis; dna; double stranded dna; 5' untranslated region; nucleic acids; biological activities; in-vivo; crystal structure; threonine; 3' untranslated region; pluripotent; kinetic analysis; adenosine triphosphate; polydeoxyribonucleotide synthase; in-vitro; methionine; lysine; synthesis (chemical); dna substrates; arginine; phenylalanine; chlorella virus; dna ligases; c-shaped; protein clamps; structural module; binding residues; adenosine phosphate; adenylation; minor grooves; dna conformation; dna template; dna strands; viruses; adenylylation; phosphodiesters; biochemical tests; induced fit; sensing capacity; sensing functions; three component; dna footprinting |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 286 |
| Issue: | 15 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2011-04-15 |
| Start Page: | 13314 |
| End Page: | 13326 |
| Language: | English |
| DOI: | 10.1074/jbc.M111.226191 |
| PROVIDER: | scopus |
| PMCID: | PMC3075678 |
| PUBMED: | 21335605 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 23 June 2011" - "CODEN: JBCHA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work