DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities.

J.W George,S.W Matson

doi:10.1016/s0021-9258(18)61619-9

Abstract

Escherichia coli helicase II has been purified to near homogeneity from cells harboring a multicopy plasmid containing the structural gene for helicase II, uvrD. In this paper a detailed description of the single-stranded DNA-dependent nucleoside 5'-triphosphatase and helicase reactions catalyzed by helicase II is presented. The results of this study suggest that nucleoside 5'-triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA. Measurements of the rate of ATP hydrolysis using a variety of single-stranded DNAs of known structure and length suggest a processive translocation mechanism for helicase II. Single-stranded DNA coated with either Escherichia coli single-stranded DNA binding protein (SSB) or bacteriophage T4 gene 32 protein fails to support helicase II ATPase activity. Moreover, helicase II is apparently unable to displace a molecule of bound SSB protein from single-stranded DNA when it is encountered in the process of translocation along a single-stranded DNA effector. The helicase reaction has been characterized using an in vitro strand displacement helicase assay. The helicase reaction requires concomitant nucleoside 5'-triphosphatase hydrolysis that is satisfied by the hydrolysis of either rATP or dATP. As the length of duplex DNA present in the partial duplex helicase substrate is increased from 71 base pairs to 343 base pairs, the fraction of duplex DNA molecules that are unwound by helicase II decreases in the absence of any accessory proteins. However, the total number of base pairs of duplex DNA unwound depends primarily on the amount of enzyme added to the helicase reaction and not on the length of the duplex DNA present in the partial duplex DNA substrate. These data suggest the number of base pairs of duplex DNA unwound is directly proportional with the concentration of helicase II in the reaction mixture. In addition, the rate of the unwinding reaction is independent of the length of the duplex DNA available for unwinding. Helicase II has been shown to dissociate from single-stranded DNA molecules infrequently acting as an ATPase. However, the enzyme dissociates from partial duplex helicase substrates more frequently. This suggests a more distributive reaction mechanism on duplex DNA than was observed on single-stranded DNA substrates. The fraction of 343-base pair partial duplex DNA molecules unwound by helicase II can be increased by the addition of appropriate concentrations of E. coli SSB to the reaction. This suggests that helicase II and SSB may act in a concerted reaction to unwind duplex DNA.

Highlights

The results of this study suggest that nucleoside 5‘triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA
When ATP(-y)S is added to a helicase reaction mixture containing ATP there is an inhibition of the helicase reaction. These results suggest that helicase I1 requires concomitant ATP hydrolysis to catalyze unwinding of the partial duplex DNA substrate
One reason for an excessof helicase IT molecules compared with DNA substrate molecules is the fact that helicase I1 interacts with the duplex DNA in a protein concentration dependent reaction

Summary

Methods

Helicase Substrate Preparation-The DNA substrate used in helicase assays consists of the 32P-labeledcomplementary strand of a restriction fragment that has been annealed to single-stranded M13 phage DNA to create a partiadluplex. The resulting partial duplex was labeled at the 3'-OH terminusina reaction mixture containing 40 mM Tris-HC1 (pH 7.5), 10mM MgCl,, 50 mM NaC1, 1 mM dithiothreitol, 40-80 pCi [ C Y - ~ ~ P I ~aCnTd P5, units of DNA polymerase I (large fragment). Unreacted radioactive nucleotide and thecomplementary strand of the restriction fragment (which does notanneal to M13mp[7] single-stranded DNA) were removed bygel filtration through a 1 ml Sepharose 6B or agarose A5M (Bio-Rad) column eluted with 10 mM Tris-HC1 (pH 7.5),0.5 mM EDTA, 100 mM NaCl. The void volume, containing the 3'-endlabeled substrate, ispooled and used directly in helicase assays. Reaction mixtures (20 pl) contained 40 mM Tris-HC1 (pH 7.5), 4 mM MgCl,, 1 mM dithiothreitol, 50 pg/ml bovine serum albumin, 1.8 mM ATP (unless otherwise indicated),and approximately 2 p~ substrate DNA.

RESULTS

Findings

DISCUSSION