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AssigneeSloan-Kettering Institute for Cancer Research
InventorsStewart Shuman, Verl Sriskanda
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This invention provides methods for the discovery of molecules that target an essential step of bacterial DNA replication—the sealing of DNA strands by NAD+-dependent DNA ligase. Specific structural components of NAD+-dependent DNA ligase that are important for the reaction of DNA ligase with NAD+ and that comprise a putative binding site for the NMN component of NAD+ were identified. The invention also includes recombinant DNA ligase enzymes that are defective in their reaction with NAD+, but active in the ligation of pre-adenylated DNA nicks. An underlying principle of this invention is the use of NAD+-reactive and NAD+-defective ligases to identify molecules that specifically bind to the NAD+-binding site of DNA ligase and thereby interfere with the reaction of DNA ligase with NAD+.
An isolated NAD+-dependant DNA ligase enzyme that is defective in its reaction with NAD+ but is active in the ligation of pre-adenylated DNA nicks, wherein said DNA ligase is the Amsacta moorei entomopoxvirus NAD+-dependent DNA ligase, wherein said DNA ligase is mutated by substituting one or more conserved residues selected from the group consisting of Tyrosine-39, Tyrosine-40, Aspartate-48, Tyrosine-51 and Aspartate-52 in domain Ia of the said DNA ligase wherein said conserved residues correspond to amino acid positions 21, 22, 30, 33 and 34 of SEQ. ID. NO:
An isolated NAD+-dependant DNA ligase enzyme that is defective in its reaction with NAD+ but is active in the ligation of pre-adenylated DNA nicks, wherein said DNA ligase is the Escherichia coli NAD+-dependent DNA ligase, wherein said DNA ligase is mutated by substituting one or more conserved residues selected from the group consisting of Tyrosine-22, Histidine-23, Aspartate-32, Tyrosine-35 and Aspartate-36 in domain Ia of the said DNA ligase wherein said conserved residues correspond to amino acid positions 21, 22, 31, 34 and 35 of SEQ. ID. NO:
Dependencies are inferred from the claim text.