Linked Life Data

8496164

Source:http://linkedlifedata.com/resource/pubmed/id/8496164

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
15
pubmed:dateCreated
1993-6-24
pubmed:abstractText
Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit. Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity. The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity. Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat. Thus, the effect of these conserved mutations is manifest in altered rates of catalysis. Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding. Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal. The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis. The Asp1002 may participate directly in chelating the metal. Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion. In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP. Models of the involvement of region I in catalysis and aphidicolin interaction are proposed. The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
25
pubmed:volume
268
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
11028-40
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:8496164-Amino Acid Sequence, pubmed-meshheading:8496164-Animals, pubmed-meshheading:8496164-Aphidicolin, pubmed-meshheading:8496164-Aspartic Acid, pubmed-meshheading:8496164-Baculoviridae, pubmed-meshheading:8496164-Cell Line, pubmed-meshheading:8496164-Cloning, Molecular, pubmed-meshheading:8496164-DNA Polymerase II, pubmed-meshheading:8496164-DNA Replication, pubmed-meshheading:8496164-Deoxyribonuclease I, pubmed-meshheading:8496164-Escherichia coli, pubmed-meshheading:8496164-Humans, pubmed-meshheading:8496164-Hydrogen Bonding, pubmed-meshheading:8496164-Kinetics, pubmed-meshheading:8496164-Magnesium, pubmed-meshheading:8496164-Manganese, pubmed-meshheading:8496164-Moths, pubmed-meshheading:8496164-Mutagenesis, Site-Directed, pubmed-meshheading:8496164-Plasmids, pubmed-meshheading:8496164-Recombinant Proteins, pubmed-meshheading:8496164-Restriction Mapping, pubmed-meshheading:8496164-Templates, Genetic, pubmed-meshheading:8496164-Transfection
pubmed:year
1993
pubmed:articleTitle
Mutational analysis of the human DNA polymerase alpha. The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis.
pubmed:affiliation
Department of Pathology, Stanford University School of Medicine, California 94305.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.