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

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 Angstrom (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 a 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.
Publisher
PORTLAND PRESS
Issue Date
1998-09
Language
ENG
Keywords

SINGLE-STRANDED-DNA; LARGE TUMOR-ANTIGEN; SACCHAROMYCES-CEREVISIAE; BINDING PROTEIN; REPLICATION PROTEIN; VIRAL ORIGIN; CALF THYMUS; T-ANTIGEN; PURIFICATION; GENE

Citation

BIOCHEMICAL JOURNAL, v.334, no.2, pp.386 - 2

ISSN
0264-6021
URI
http://hdl.handle.net/10203/74835
Appears in Collection
BS-Journal Papers(저널논문)
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