The Base Pairing Partner Modulates Alkylguanine Alkyltransferase.

O6-Alkylguanine DNA adducts are repaired by the suicide enzyme alkylguanine alkyltransferase (AGT). AGT facilitates repair by binding DNA in the minor groove, flipping out the damaged base, and transferring the O6-alkyl group to a cysteine residue in the enzyme's active site. Despite there being significant knowledge concerning the mechanism of AGT repair, there is limited insight regarding how altered interactions of the adduct with its complementary base in the DNA duplex influence its recognition and repair. In this study, the relationship of base pairing interactions and repair by human AGT (hAGT) was tested in the frequently mutated codon 12 of the KRAS gene with complementary sequences containing each canonical DNA base. The rate of O6-MeG repair decreased 2-fold when O6-MeG was paired with G, whereas all other canonical bases had no impact on the repair rate. We used a combination of biochemical studies, molecular modeling, and artificial nucleobases to elucidate the mechanism accounting for the 2-fold decrease. Our results suggest that the reduced rate of repair is due to O6-MeG adopting a syn conformation about the glycosidic bond precluding the formation of a repair-active complex. These data provide a novel chemical basis for how direct reversion repair may be impeded through modification of the base pair partner and support the use of artificial nucleobases as tools to probe the biochemistry of damage repair processes.

Mechanism of RNA polymerase II stalling by DNA alkylation.

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

Minor Groove 3-Deaza-Adenosine Analogues: Synthesis and Bypass in Translesion DNA Synthesis.

Anticancer drugs that alkylate DNA in the minor groove may give rise to 3-alkyl-adenosine adducts that interfere with replication, inducing apoptosis in rapidly dividing cancer cells. However, translesion DNA synthesis (TLS) by polymerase enzymes (Pols) with the capacity to bypass DNA adducts may contribute to damage tolerance and drug resistance. 3-Alkyl-adenosine adducts are unstable and depurinate, which is a barrier to addressing chemical and enzymatic aspects of how they impact the progress of DNA Pols. To characterize structure-based relationships of 3-adenine alkylation relevant to cancer drugs on duplex stability and DNA Pol-catalyzed DNA synthesis, we synthesized stable 3-deaza-3-alkyl-adenosine analogues, including 3-deaza-3-phenethyl-adenosine and 3-deaza-3-methoxynaphthylethyl-adenosine, and incorporated them into oligonucleotides. A moderate reduction of duplex stability was observed on the basis of thermal denaturation data. Replication studies using purified Y-family human DNA Pols hPol η, κ, and ι indicated that these enzymes can perform TLS over the modified bases. hPol η had higher misincorporation rates when synthesizing opposite the modified bases compared with adenine, whereas hPol κ and ι maintained high fidelity. These results provide insight into how alterations in chemical structure reduce bypass of minor-groove adducts, and provide novel chemical probes for evaluating minor-groove DNA alkylation.

Generation of DNA interstrand cross-links by post-synthetic reductive amination.

DNA interstrand cross-links (ICLs) are the clinically most relevant adducts formed by many antitumor agents. To facilitate the study of biological responses triggered by ICLs, we developed a new approach toward the synthesis of mimics of nitrogen mustard ICLs. 7-Deazaguanine residues bearing acetaldehyde groups were incorporated into complementary strands of DNA and cross-link formation induced by double reductive amination. Our strategy enables the synthesis of major groove cross-links in high yields and purity.