Nucleic Acids Research, Vol 24, Issue 11 2087-2094, Copyright © 1996 by Oxford University Press
K Bender, M Federwisch, U Loggen, P Nehls and MF Rajewsky
Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O6-
ethylguanine (O6-EtGua) flanked 5' and 3' by different bases (5'..TGT..3';
5'..CGG..3', 5'..GGT..3'; 5'..GGG..3'; 5'..GGA..3') were synthesized to
investigate the binding and repair characteristics of recombinant human
O6-alkylguanine-DNA alkyltransferase (AT) in vitro. The apparent
association constant (KA(app)) of AT to the oligomers and the repair rate
constant for O6-EtGua (k) respectively, were determined by gel retardation
and a monoclonal antibody-based filter binding assay. When ds- or
single-stranded (ss) oligomers with or without O6- EtGua were used, no
major differences in KA(app) values were observed with either substrate:
KA(app) values for native AT were 7.1 and 8.4 x 10(5) M(-1) respectively,
for unmodified and [O6-EtGua]-containing ds- oligomers. The corresponding
values for ss-oligomers were 1.0 and 4.9 x 10(5) M(-1). The N-terminal
first 56 amino acids of AT only exert a limited influence on DNA binding;
the KA(app) values for an N- terminally truncated AT protein (1.1 x 10(5)
M(-1)) and native AT were of the same order. Moreover, KA(app) was hardly
affected by Cys(145)- methylated AT (2.0 x 10(5) M(-1)). The k-values
(6.5-11.5 x 10(6) M(- 1)s(-1)) were not significantly dependent on
nucleotide sequence. k- values of 5.3 and 4.0 x 10(6) M(-1)s(-1)
respectively, were obtained with the N-terminally truncated AT protein and
for repair of the postreplicative mispair [O6-EtGua]: T by native AT. The
low KA(app), the negligible influence on O6 of ethylation, and the minor
modulation KA(app) and k by varying the bases flanking O6-EtGua, all
indicate that the binding of AT to DNA is non-specific and mediated mainly
by ionic interactions [reduced KA(app) and k-values at increased ionic
strength]. Surplus DNA reduces the rate of O6-EtGua repair in ds- oligomers
by competitive binding of AT molecules. The reaction mechanism of AT with
DNA in vivo requires further investigation.
ARTICLES
Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context
Institute of Cell Biology (Cancer Research), University of Essen Medical School, Germany.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. J. Rasimas, S. R. Kar, A. E. Pegg, and M. G. Fried Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs J. Biol. Chem., February 2, 2007; 282(5): 3357 - 3366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zang, Q. Fang, A. E. Pegg, and F. P. Guengerich Kinetic Analysis of Steps in the Repair of Damaged DNA by Human O6-Alkylguanine-DNA Alkyltransferase J. Biol. Chem., September 2, 2005; 280(35): 30873 - 30881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Rasimas, A. E. Pegg, and M. G. Fried DNA-binding Mechanism of O6-Alkylguanine-DNA Alkyltransferase. EFFECTS OF PROTEIN AND DNA ALKYLATION ON COMPLEX STABILITY J. Biol. Chem., February 28, 2003; 278(10): 7973 - 7980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. E. Verdemato, J. A. Brannigan, C. Damblon, F. Zuccotto, P. C. E. Moody, and L.-Y. Lian DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase Nucleic Acids Res., October 1, 2000; 28(19): 3710 - 3718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Singer and B. Hang Nucleic acid sequence and repair: role of adduct, neighbor bases and enzyme specificity Carcinogenesis, June 1, 2000; 21(6): 1071 - 1078. [Full Text] [PDF]
|
|
|
|
|
|
B. Hang, J. Sagi, and B. Singer Correlation between Sequence-dependent Glycosylase Repair and the Thermal Stability of Oligonucleotide Duplexes Containing 1,N6-Ethenoadenine J. Biol. Chem., December 11, 1998; 273(50): 33406 - 33413. [Abstract] [Full Text] [PDF]
|
|