We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.
The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least =2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.The Escherichia coli helicase II protein, the product of the uvrD gene (1-4), is a DNA-dependent ATPase and helicase (5, 6), which catalyzes the unwinding of duplex DNA with an apparent 3' to 5' polarity (7). Helicase II has been implicated to function in replication (8), as well as recombination (9-11). This protein is also required in methyl-directed mismatch (12-14) and uvrABC excision repair pathways (15-17), both of which require a nicked DNA as an intermediate. In methyl-directed mismatch repair, helicase II protein has been proposed to initiate DNA unwinding at the mismatch (18), although initiation at the nick has not been ruled out. Most previous studies of DNA unwinding by helicase II in vitro have concluded that a flanking region of single-stranded DNA (ssDNA), 3' to the duplex, is required to initiate unwinding (7,19 MATERIALS AND METHODSBuffers and Enzymes. Buffers were made with distilled deionized Milli-Q water or, for EM, two times glass-distilled water that was redistilled within 12 hr of use. TE buffer is 10 mM Tris-HCl (pH 8.1)/1 mM Na3EDTA. Unwinding buffer (pH 7.5 at 370C) is 40 mM Hepes'KOH/0.1 mM dithiothreitol/1.5 mM ATP/0.5 mM MgCl2.Helicase II protein was purified from E. coli N4830/ pTLS1, and its concentration was determined spectrophotometrically (20). Helicase II protein stocks were tested for exonuclease activity on both 5' and 3' end-labeled ss-and duplex DNA under the same conditions used in the unwinding experiments. Helicase II and 32P end-labeled DNA were incubated at 370C and then subjected to electrophoresis on polyacrylamide gels. After 45 min, <0.4% of the 32p label had been removed from the ends of the DNA substrates. fi gene II protein was purified from E. coli K561/pDG117IIA (21), according to S. Johnston and D. Ray (University of California at Los Angeles) with some modifications.DNA Substrates. M13mpll replicative form (RF) DNA and pUC8 DNA were isolated as described (...
Escherichia coli helicase II (UvrD) protein can initiate unwinding of duplex DNA at blunt ends or nicks, although these reactions require excess protein. We have undertaken kinetic studies of these reactions in order to probe the mechanism of initiation of unwinding. DNA unwinding was monitored directly by using agarose gel electrophoresis and indirectly through the rate of ATP hydrolysis by helicase II in the presence of an ATP-regenerating system. In the presence of fully duplex DNA and excess helicase II, the rate of ATP hydrolysis displays a distinct lag phase before the final steady-state rate of hydrolysis is reached. This reflects the fact that ATP hydrolysis under these conditions results from helicase II binding to the ssDNA products of the unwinding reaction, rather than from an intrinsic duplex DNA-dependent ATPase activity. Unwinding of short blunt-ended duplex DNA (341 and 849 base pairs) occurs in an "all-or-none" reaction, indicating that initiation of unwinding by helicase II is rate-limiting. We propose a minimal mechanism for the initiation of DNA unwinding by helicase II which includes a binding step followed by the rate-limiting formation of an initiation complex, possibly involving protein dimerization, and we have determined the phenomenological kinetic parameters describing this mechanism. Unwinding of a series of DNA substrates containing different initiation sites (e.g., blunt ends, internal nicks, and four-nucleotide 3' vs 5' ssDNA flanking regions) indicates that the rate of initiation is slowest at nicks and, surprisingly, at ends possessing a four-nucleotide 3' ssDNA flanking region.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.