Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines.
A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon >> 3-carbon > 4-carbon.
Kowalczyk
Agnieszka
A
Chemistry Department and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Carmical
J Russ
JR
Zou
Yue
Y
Van Houten
Bennett
B
Lloyd
R Stephen
RS
Harris
Constance M
CM
Harris
Thomas M
TM
D001483:Base Sequence; D003432:Cross-Linking Reagents; D018736:DNA Adducts / Q000138:chemical synthesis / Q000737:chemistry*; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D006147:Guanine / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D006147:Guanine; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC