Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Neoplasias Cutâneas/patologiaRESUMO
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.
Assuntos
Códon/genética , Adutos de DNA/metabolismo , Reparo do DNA , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Pareamento de Bases , Códon/metabolismo , Adutos de DNA/genética , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Modelos Moleculares , Mutação , TermodinâmicaRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/química , RNA Polimerase II/química , Sesquiterpenos/farmacologia , Compostos de Espiro/farmacologia , Antineoplásicos Alquilantes/química , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , DNA de Neoplasias/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Cinética , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Sesquiterpenos/química , Compostos de Espiro/químicaRESUMO
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.
Assuntos
Adenosina/análogos & derivados , DNA/metabolismo , Adenosina/síntese química , Adenosina/metabolismo , Pareamento de Bases , Sítios de Ligação , Domínio Catalítico , DNA/química , Adutos de DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Temperatura de Transição , DNA Polimerase iotaRESUMO
Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O(6)-G alkylation adducts. To optimize O(6)-Me-G-targeting probes, an understanding of how base pairs with O(6)-Me-G are stabilized is needed. In this study, we compared the ability of O(6)-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O(6)-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O(6)-G alkylation adducts in DNA.
Assuntos
Adutos de DNA/química , DNA/química , Sondas de Oligonucleotídeos/química , Oligonucleotídeos/química , Pirimidinas/química , Ligação de Hidrogênio , TermodinâmicaRESUMO
O6-Methylguanine (O6-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O6-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O6-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O6-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O6-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O6-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O6-MeG without a significant loss of efficiency.
Assuntos
DNA Polimerase beta/metabolismo , Corantes Fluorescentes/química , Guanina/análogos & derivados , Nucleosídeos/química , Sulfolobus solfataricus/enzimologia , Corantes Fluorescentes/metabolismo , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Nucleosídeos/metabolismoRESUMO
The missing linking: BCNU is a chemotherapy drug that generates an ethylene bridge between N(1) of deoxyguanosine and N(3) of deoxycytidine. No synthesis of a DNA containing this moiety has been reported until now. A new strategy uses a photocaged nucleobase that, when released, generates a highly reactive intermediate which cross-links the opposing DNA strand in a manner analogous to BCNU.
Assuntos
Carmustina/química , Reagentes de Ligações Cruzadas/química , DNA/química , Alquilação , Desoxicitidina/química , Desoxiguanosina/químicaRESUMO
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.
Assuntos
Antineoplásicos/química , Reagentes de Ligações Cruzadas/química , DNA/química , Modelos Moleculares , Oligonucleotídeos/síntese química , Aminação , Mecloretamina/química , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.