Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA

Biochemistry ◽  
1993 ◽  
Vol 32 (30) ◽  
pp. 7765-7771 ◽  
Author(s):  
Jaya G. Yodh ◽  
Floyd R. Bryant
Keyword(s):  
Atp Hydrolysis ◽  
Dna Helicase ◽  
Duplex Dna ◽  
Dna Helicase Ii ◽  
Kinetics Of

scholarly journals Identification and Characterization ofEscherichia coli DNA Helicase II Mutants That Exhibit Increased Unwinding Efficiency

1998 ◽  
Vol 180 (2) ◽  
pp. 377-387 ◽  
Author(s):  
Gang Zhang ◽  
Enxin Deng ◽  
Larry Baugh ◽  
Sidney R. Kushner
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Atp Hydrolysis ◽  
Specific Activity ◽  
Dna Helicase ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Biochemical Basis ◽  
Dna Helicase Ii

ABSTRACT Using a combination of both ethyl methanesulfonate and site-directed mutagenesis, we have identified a region in DNA helicase II (UvrD) from Escherichia coli that is required for biological function but lies outside of any of the seven conserved motifs (T. C. Hodgman, Nature 333:22–23, 1988) associated with the superfamily of proteins of which it is a member. Located between amino acids 403 and 409, alterations in the amino acid sequence DDAAFER lead to both temperature-sensitive and dominant uvrDmutations. The uvrD300 (A406T) and uvrD301(A406V) alleles produce UV sensitivity at 44°C but do not affect sensitivity to methyl methanesulfonate (MMS). In contrast, theuvrD303 mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is dominant to uvrD+ when present at six to eight copies per cell. Several of the alleles demonstrated a strong antimutator phenotype. In addition, conjugal recombination is reduced 10-fold in uvrD303 strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (uvrD302) was neutral. To determine the biochemical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a uvrDΔ294 deletion background and characterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild-type control, while itsKm for ATP binding remains unchanged. More importantly, the UvrD303 protein unwinds partial duplex DNA up to 10 times more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propose that the region located between amino acids 403 and 409 serves to regulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA repair systems in which the protein participates.

scholarly journals Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5′-to-3′ direction

1998 ◽  
Vol 334 (2) ◽  
pp. 377-386 ◽  
Author(s):  
Changwoo LEE ◽  
Yeon-Soo SEO
Keyword(s):  
Atpase Activity ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Atp Hydrolysis ◽  
Sedimentation Coefficient ◽  
Dna Helicase ◽  
Single Stranded Dna ◽  
Dna Helicase Ii ◽  
Isolation And Characterization

We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe. The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity. Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel. The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 Å (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa. The enzyme translocates in a 5´-to-3´ direction with respect to the substrate strand to which it is bound. Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP. This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP. Non-hydrolysable ATP analogues did not support helicase activity. Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50–100 s of incubation. Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding. The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor. In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5–10 min in the presence of shorter oligonucleotides. The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used. These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive.

scholarly journals Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

1994 ◽  
Vol 13 (20) ◽  
pp. 4991-5001 ◽  
Author(s):  
N. Tuteja ◽  
R. Tuteja ◽  
A. Ochem ◽  
P. Taneja ◽  
N.W. Huang ◽  
...  
Keyword(s):  
Dna Helicase ◽  
Dna Unwinding ◽  
Dna Helicase Ii ◽  
Human Dna ◽  
Ku Autoantigen

Transient kinetics of ATP hydrolysis by covalently crosslinked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method

Biochemistry ◽  
1985 ◽  
Vol 24 (14) ◽  
pp. 3814-3820 ◽  
Author(s):  
J. A. Biosca ◽  
F. Travers ◽  
T. E. Barman ◽  
R. Bertrand ◽  
E. Audemard ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Atp Hydrolysis ◽  
Transient Kinetics ◽  
Actomyosin Complex ◽  
Rapid Flow ◽  
Kinetics Of ◽  
Quench Method

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

scholarly journals Stimulation of the UvrABC enzyme-catalyzed repair reactions by the UvrD protein (DNA helicase II)

1985 ◽  
Vol 13 (5) ◽  
pp. 1483-1492 ◽  
Author(s):  
Keiko Kumura ◽  
Mutsuo Sekiguchi ◽  
Anne-Lill Stemum ◽  
Erling Seeberg
Keyword(s):  
Dna Helicase ◽  
Dna Helicase Ii ◽  
Stimulation Of

scholarly journals Actinomycin D Induces Histone γ-H2AX Foci and Complex Formation of γ-H2AX with Ku70 and Nuclear DNA Helicase II

2005 ◽  
Vol 280 (10) ◽  
pp. 9586-9594 ◽  
Author(s):  
Hannah Elisabeth Mischo ◽  
Peter Hemmerich ◽  
Frank Grosse ◽  
Suisheng Zhang
Keyword(s):  
Complex Formation ◽  
Actinomycin D ◽  
Nuclear Dna ◽  
Dna Helicase ◽  
Dna Helicase Ii
Sign in / Sign up

Export Citation Format

Share Document