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Volume 271, Number 41, Issue of October 11, 1996 pp. 25360-25368
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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A Partially Functional DNA Helicase II Mutant Defective in Forming Stable Binary Complexes with ATP and DNA
A ROLE FOR HELICASE MOTIF III

(Received for publication, May 10, 1996, and in revised form, July 24, 1996)

Robert M. Brosh Jr. and Steven W. Matson ^§

From the Department of  Biology and the ^§ Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent K_m for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase.

The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.

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