Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.25 (triphosphatase)
 1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five.
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PMID:Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli. 282 20

Escherichia coli helicase II, product of the uvrD gene, is a single-stranded DNA-dependent nucleoside 5'-triphosphatase with helicase activity. As a DNA-dependent ATPase, helicase II translocates processively along single-stranded DNA (S. W. Matson, unpublished results). The direction of translocation has been determined using a helicase assay that directly measures the ability of helicase II to catalyze the displacement of a labeled DNA fragment from one end of a single-stranded linear DNA molecule. The translocation of helicase II along single-stranded DNA is unidirectional and in the 3' to 5' direction with respect to the DNA strand on which the enzyme is bound. A kinetic analysis of the displacement of a labeled DNA fragment annealed to a linear single-stranded DNA molecule is also consistent with unidirectional translocation in the 3' to 5' direction. These results are contrary to results previously obtained using an indirect helicase assay (Kuhn, B., Abdel-Monem, M., Krell, H., and Hoffmann-Berling, H. (1979) J. Biol. Chem. 254, 11343-11350).
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PMID:Escherichia coli helicase II (urvD gene product) translocates unidirectionally in a 3' to 5' direction. 294 37

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.
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PMID:DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities. 302 63