Linked Life Data

12966083

Source:http://linkedlifedata.com/resource/pubmed/id/12966083

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
47
pubmed:dateCreated
2003-11-17
pubmed:abstractText
Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
21
pubmed:volume
278
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
46994-7001
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site.
pubmed:affiliation
Department of Pathology, University of Washington, 1959 NE Pacific Avenue, Seattle, WA 98195-7705, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.