Effect of cross-link structure on DNA interstrand cross-link repair synthesis.

DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link.

Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase.

O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand.

Synthesis, biophysical and repair studies of O6-2'-deoxyguanosine adducts by Escherichia coli OGT.

Oligonucleotides containing modified 2'-deoxyguanosines bearing a seven carbon linker at the O(6)- atom with either a terminal hydroxyl or 2'- deoxyguanosine group have been synthesized as potential intermediates formed during repair of interstrand cross-linked DNA. Repair of these substrates with Escherichia coli OGT was investigated with an assay involving cleavage of the unmodified duplex with the restriction endonuclease PvuII followed by analysis of the products by denaturing polyacrylamide gel electrophoresis. Duplexes containing these modifications were repaired by OGT suggesting that direct repair may play a role, in combination with other repair pathways, in reversing interstrand crosslink DNA damage.

Effect of linker length on DNA duplexes containing a mismatched O6-2'-deoxyguanosine-alkyl interstrand cross-link.

DNA duplexes containing a directly opposed O(6)- alkyl-2'-deoxyguanosine interstrand cross-link were synthesized to serve as structural mimics of lesions formed by the bifunctional chemotherapeutic alkylating agents busulfan and hepsulfam. One of the key steps to prepare the necessary bis-phosphoramidites involved the Mitsunobu reaction between a diol linking two protected 2'-deoxyguanosine nucleosides at the O(6) position. These bis-phosphoramidites were incorporated into 11-bp DNA duplexes by solid phase synthesis to produce cross-linked DNA probes in high yields.