Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency.

Double-stranded DNA needs to be unwound in order for replication, repair, and recombination to proceed. A class of enzymes, designated DNA helicases, accomplish this task by using the energy of nucleoside 5′-triphosphate hydrolysis to disrupt the hydrogen bonds between the Watson-Crick base pairs of duplex DNA. There are at least 12 helicases in Escherichia coli (24, 25). One of these, DNA helicase II, the product of the uvrD gene, has been demonstrated to play a role in at least two distinct DNA repair pathways: nucleotide excision repair and methyl-directed mismatch repair (29, 39). Analysis of more than a dozen mutant alleles of uvrD has suggested that DNA helicase II may also be involved in homologous recombination and replication (5, 12, 53). DNA helicase II is a single-stranded DNA-dependent ATPase that unwinds double-stranded DNA with a 3′-to-5′ polarity with respect to the DNA strand on which the enzyme initially binds (23). At low protein concentrations, DNA helicase II preferentially unwinds duplex DNA possessing 3′-flanking single-stranded DNA. At much higher concentrations of DNA helicase II, unwinding of blunt-ended or nicked duplex DNA is observed (37). While it has been reported that DNA helicase II is monomeric in solution (1, 35), dimers or oligomers can be formed upon binding DNA (20), and it is proposed that the dimeric helicase is the functionally active form in DNA unwinding (3). Computer analysis of the uvrD coding sequence has indicated that it contains seven conserved motifs that are shared by a superfamily of proteins involved in DNA metabolism (16). Motifs IA, IB, and II, previously identified as Walker A and Walker B sequences, respectively, are conserved segments found in many nucleoside triphosphate-binding proteins and all helicases characterized to date (14, 15, 46). Mutations within these two motifs of DNA helicase II result in proteins with significantly reduced ATP-binding and/or ATP hydrolysis activity (8, 12, 48). In addition, there is genetic evidence that all of the remaining motifs (III to VI) are also required for the biological functions of DNA helicase II (8–10, 51). While study of the seven conserved motifs can lead to a better understanding of the universal structure-function relationships among this family of proteins, identification of important regions unique to UvrD could help better explain the specialization among DNA helicases and provide a deeper insight into the specific roles of DNA helicase II in vivo. For example, while DNA helicase II and the Rep helicase share 40% homology at the amino acid level (13), the Rep enzyme works catalytically (4), while the UvrD protein is required in stoichiometric amounts (2, 17). In this communication, we report the identification of a new region of DNA helicase II that is required for in vivo function. Located between amino acids 403 and 409, mutations in this region lead to both temperature-sensitive and dominant phenotypes. Among the five mutant alleles identified, the substitution of two adjacent aspartic acid residues with alanines (uvrD303) resulted in a dominant negative phenotype for UV and methyl methanesulfonate (MMS) sensitivity while exhibiting reduced levels of spontaneous mutagenesis and homologous recombination. The purified UvrD303 protein exhibits a higher specific activity for DNA-dependent ATP hydrolysis and unwinds DNA partial duplexes up to 10 times more efficiently than the wild-type DNA helicase II protein. The implications of these results are discussed.