INVITRO MUTAGENESIS AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI OF A CDNA-ENCODING THE CATALYTIC DOMAIN OF HUMAN DNA LIGASE-I

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Abstract
Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E.coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E.coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E.coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.