Linked Life Data

9730821

Source:http://linkedlifedata.com/resource/pubmed/id/9730821

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
36
pubmed:dateCreated
1999-2-5
pubmed:abstractText
Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
8
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12489-95
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:9730821-Alanine, pubmed-meshheading:9730821-Amino Acid Sequence, pubmed-meshheading:9730821-DNA Repair, pubmed-meshheading:9730821-DNA-Binding Proteins, pubmed-meshheading:9730821-Enzyme Activation, pubmed-meshheading:9730821-Glutamic Acid, pubmed-meshheading:9730821-Guanine, pubmed-meshheading:9730821-Humans, pubmed-meshheading:9730821-Kinetics, pubmed-meshheading:9730821-Molecular Sequence Data, pubmed-meshheading:9730821-Mutagenesis, Site-Directed, pubmed-meshheading:9730821-O(6)-Methylguanine-DNA Methyltransferase, pubmed-meshheading:9730821-Oligodeoxyribonucleotides, pubmed-meshheading:9730821-Phenylalanine, pubmed-meshheading:9730821-Protein Conformation, pubmed-meshheading:9730821-Recombinant Proteins, pubmed-meshheading:9730821-Threonine, pubmed-meshheading:9730821-Tyrosine
pubmed:year
1998
pubmed:articleTitle
Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.
pubmed:affiliation
Department of Cellular and Molecular Physicology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.