Linked Life Data

9341218

Source:http://linkedlifedata.com/resource/pubmed/id/9341218

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
43
pubmed:dateCreated
1997-11-17
pubmed:abstractText
We have cloned and expressed the yeast DNA helicase A in Escherichia coli at a high level (approximately 30 mg/L of culture) in soluble form. We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electrophoresis the enzyme behaved as a approximately 90 kDa protein. The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt (NaCl) dependent. In low-salt buffers (containing 50 mM NaCl), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure. In higher salt buffer (containing greater than 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze ATP or dATP with equal efficiency. The ATPase activity of the enzyme was absolutely DNA-dependent. The nucleotidase activities were comparable to those of the native enzyme. Kinetic analysis of the ATPase activity demonstrated that the Km of the enzyme was approximately 90 microM and the rate of ATP hydrolysis was approximately 20 ATP s-1 molecule-1. DNA sequences containing pyrimidine stretches were more effective activators than those containing purine stretches. However, poly(dC) appeared to be the most effective activator of the ATPase activity. The ATPase activity was inhibited by salt (NaCl) above 50 mM with a half-maximal inhibition at approximately 110 mM. It is likely that the active state of helicase A is hexameric. The helicase activity of the recombinant enzyme was stimulated significantly by the yeast replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of E. coli (SSB). The DNA helicase migrated on a DNA template in a 5' --> 3' direction. Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/SSB, and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
13277-84
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization.
pubmed:affiliation
Department of Molecular Biology, Science Center, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.