Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II

1995 ◽  
Vol 146 (2) ◽  
pp. 121-126 ◽  
Author(s):  
Jamboor K. Vishwanatha ◽  
Thomas J. Tauer ◽  
Solon L. Rhode
Keyword(s):  
Hela Cell ◽  
Dna Helicase ◽  
Single Stranded Dna ◽  
Dna Helicase Ii ◽  
Dependent Atpase

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 A Region Near the C-Terminal End ofEscherichia coli DNA Helicase II Is Required for Single-Stranded DNA Binding

1999 ◽  
Vol 181 (8) ◽  
pp. 2519-2526 ◽  
Author(s):  
Leah E. Mechanic ◽  
Marcy E. Latta ◽  
Steven W. Matson
Keyword(s):  
Dna Binding ◽  
Excision Repair ◽  
Dna Helicase ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Dna Helicase Ii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Apparent Homogeneity ◽  
C Terminus

ABSTRACT The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDΔ107C (deletion of the last 107 C-terminal amino acids), UvrDΔ102C, and UvrDΔ40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDΔ107C and UvrDΔ102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDΔ40C protein fully complemented the loss of helicase II in both repair pathways. UvrDΔ102C and UvrDΔ40C were purified to apparent homogeneity and characterized biochemically. UvrDΔ102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (k cat = 0.01% of the wild-type level). UvrDΔ40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.

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