Intrastrand DNA cross-links as tools for studying DNA replication and repair: Two-, three-, and four-carbon tethers between the N2 positions of adjacent guanines

Agnieszka Kowalczyk, J. Russ Carmical, Yue Zou, Bennett Van Houten, R. Stephen Lloyd, Constance M. Harris, Thomas M. Harris

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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′, crosslinked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O6trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4diaminobutane): 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.

Original languageEnglish (US)
Pages (from-to)3109-3118
Number of pages10
JournalBiochemistry
Volume41
Issue number9
DOIs
StatePublished - Mar 5 2002

ASJC Scopus subject areas

  • Biochemistry

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