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Journal of Bacteriology, April 1999, p. 2519-2526, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Region Near the C-Terminal End of Escherichia coli DNA Helicase II Is Required for Single-Stranded DNA Binding

Leah E. Mechanic,1 Marcy E. Latta,2 and Steven W. Matson2,3

Department of Biochemistry and Biophysics, Protein Engineering and Molecular Genetics Training Program,1 and Department of Biology2 and Curriculum in Genetics and Molecular Biology,3 University of North Carolina, Chapel Hill, North Carolina 27599

Received 8 September 1998/Accepted 29 January 1999

The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDDelta 107C (deletion of the last 107 C-terminal amino acids), UvrDDelta 102C, and UvrDDelta 40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDDelta 107C and UvrDDelta 102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDDelta 40C protein fully complemented the loss of helicase II in both repair pathways. UvrDDelta 102C and UvrDDelta 40C were purified to apparent homogeneity and characterized biochemically. UvrDDelta 102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (kcat = 0.01% of the wild-type level). UvrDDelta 40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.

Journal of Bacteriology, April 1999, p. 2519-2526, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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