Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues

Abstract

DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.

Correlation of Thermal Stability and Structural Distortion of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites with Their Selective Formation and Repair

“…16,18 Substrates containing the cross-link with the abasic sites opposite the 3′-adjacent dT (“observed”) and with the cross-linked dA opposite the abasic site analogues (“unobserved”) were prepared. The “observed” ICLs correspond to those that are (preferentially) formed by C4-AP and DOB (Scheme 2).…”

“…15 Chemically stable analogues [X–Y and W–V (Scheme 1)] that facilitate the characterization of these reversibly formed ICLs have been prepared. 16 ICLs (and analogues) arising from oxidized abasic sites (C4-AP and DOB) are substrates for bacterial nucleotide excision repair, which transforms some of these molecules into double-strand breaks. 17,18 The chemically stabilized C4-AP ICL analogue is a poorer substrate than that resulting from DOB, but the structural basis for this difference is unknown.…”

Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases

“…The oligonucleotide containing a site-specifically modified DOB-ICL analogue was synthesized and purified as described previously. 16 The catalytic fragments of human Y-family polymerases pol ι (1–420), 33 pol η (1–432), 34 pol κ (19–526), 35 and REV1 (330–833) 36 were purified following previously published protocols; expression vectors were kindly provided by F. P. Guengerich (Vanderbilt University School of Medicine, Nashville, TN). The human DNA polymerase ν expression vector was a generous gift from R. D. Wood (University of Texas M. D. Anderson Cancer Center, Houston, TX), and the full-length recombinant pol ν was purified as described previously.…”

“…Herein, we used the previously developed strategy to synthesize an oligonucleotide containing a site-specifically modified DOB-ICL analogue. 16 This DOB-ICL models an intermediate produced by endonucleolytic incision. Using this substrate, we thoroughly characterized the bypass activities of several important TLS DNA polymerases (pols), including human pol η , pol ι , pol κ , pol ν , REV1, and yeast pol ζ .…”

O6-2′-Deoxyguanosine-butylene-O6-2′-deoxyguanosine DNA Interstrand Cross-Links Are Replication-Blocking and Mutagenic DNA Lesions

“…To understand the chemical and biological consequences of ICLs, a number of synthetic ICL substrates have been developed, which include ICLs derived from platinum drugs, nitrogen mustard, endogenous malondialdehyde (reviewed by Schärer 15 ), and different forms of abasic sites. 16,17 …”