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Nucleic Acids Research, 1991, Vol. 19, No. 22 6093-6099
© 1991

MOLECULAR BIOLOGY

In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I

Ken-ichi Kodama, Deborah E. Barnes and Tomas Lindahl*

Imperial Cancer Research Fund, Clare Hall Laboratories South Mimms, Hertfordshire EN6 3LD, UK

*To whom correspondence should be addressed

Received September 23, 1991. Accepted October 24, 1991.

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 nonpermissive 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.


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