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. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

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. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link 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. UV thermal denaturation studies revealed that these interstrand cross-linked containing oligonucleotides were stabilized compared to a DNA duplex containing a central 2'-deoxyguanosine mismatch. The duplex containing the four carbon cross-link was stabilized by 10 degrees C relative to the seven carbon linker. Molecular models of these duplexes that were geometry optimized by the AMBER force field suggest that the seven carbon cross-link was less efficiently accommodated in the major groove of the duplex relative to the four carbon linker, accounting for the observed destabilization.

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.