Mutational analysis of Chlorella virus DNA ligase: Catalytic roles of domain I and motif VI Journal Article
| Authors: | Sriskanda, V.; Shuman, S. |
| Article Title: | Mutational analysis of Chlorella virus DNA ligase: Catalytic roles of domain I and motif VI |
| Abstract: | A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs 1, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase - adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction. |
| Keywords: | mutation, missense; sequence deletion; nonhuman; dna repair; carboxy terminal sequence; dna; amino acid sequence; molecular sequence data; amino terminal sequence; kinetics; recombinant fusion proteins; dna viruses; escherichia coli; mutagenesis, site-directed; alanine; adenosine triphosphate; polydeoxyribonucleotide synthase; catalytic domain; genetic conservation; deletion mutant; chlorella virus; dna ligases; viral proteins; ligase; nucleotidyltransferase; virus dna; chlorella; priority journal; article |
| Journal Title: | Nucleic Acids Research |
| Volume: | 26 |
| Issue: | 20 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 1998-10-01 |
| Start Page: | 4618 |
| End Page: | 4625 |
| Language: | English |
| DOI: | 10.1093/nar/26.20.4618 |
| PUBMED: | 9753729 |
| PROVIDER: | scopus |
| PMCID: | PMC147904 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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