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1.
Nat Commun ; 12(1): 4671, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344863

RESUMO

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Nitrobenzenos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Tiazinas/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Nitrobenzenos/química , Nitrobenzenos/farmacologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Tiazinas/química , Tiazinas/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Commun ; 12(1): 4020, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188055

RESUMO

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'-terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.


Assuntos
Pareamento de Bases/genética , Dano ao DNA/genética , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Guanina/química , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Estresse Oxidativo/genética , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo
3.
Sci Rep ; 9(1): 16400, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704958

RESUMO

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.


Assuntos
Citarabina/farmacologia , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citarabina/análogos & derivados , Citarabina/química , Replicação do DNA/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica
4.
Sci Rep ; 8(1): 12702, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30140014

RESUMO

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2'-deoxycytidine only by the presence of an additional hydroxyl group at the C2' position of the 2'-deoxyribose. The active form of the drug AraC 5'-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3' terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3'-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.


Assuntos
Antineoplásicos/química , Citarabina/química , DNA Polimerase Dirigida por DNA/metabolismo , Antineoplásicos/farmacologia , Cristalização , Citarabina/farmacologia , Citidina/química , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Desoxicitidina/química , Humanos , Estrutura Molecular , Difração de Raios X
5.
Curr Opin Struct Biol ; 53: 77-87, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30005324

RESUMO

The eukaryotic DNA replication machinery is conserved from yeast to humans and requires the actions of multiple DNA polymerases. In addition to replicative DNA polymerases for duplication of the leading and lagging DNA strands, another group of specialized polymerases is required for DNA repair and/or translesion DNA synthesis (TLS). We emphasize here recent findings that accelerate our understanding of the structure and mechanisms of these remarkable enzymes. We also highlight growing evidence on the role of DNA polymerases in the origin of certain cancers, and paradoxically as emerging targets for cancer therapy.


Assuntos
DNA Polimerase Dirigida por DNA , DNA/metabolismo , Células Eucarióticas/enzimologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Domínios Proteicos , Estrutura Quaternária de Proteína
6.
Oncotarget ; 9(102): 37753-37765, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30701029

RESUMO

Overexpression and constitutive activation of CYCLIN D1 and Casein Kinase 2 are common features of many hematologic malignancies, including mantle cell lymphoma (MCL) and leukemias such as T-cell acute lymphoblastic leukemia (T-ALL). Although both CK2 and CDK4 inhibitors have shown promising results against these tumor types, none of these agents have achieved objective responses in the clinic as monotherapies. Because both proteins play key roles in these and other hematological malignancies, we have analyzed the therapeutic potential of ON108110, a novel dual specificity ATP-competitive inhibitor of protein kinase CK2 as well as CDK4/6 in MCL and T-ALL. We show that in cell growth inhibition assays, MCL and T-ALL cell lines exhibited increased sensitivity to ON108110 when compared to other tumor types. Treatment with ON108110 reduced the level of phosphorylated RB-family proteins. In addition, ON108110 treatment resulted in concentration dependent inhibition of PTEN phosphorylation and a concomitant decrease in PI3K-AKT signaling mediated by CK2. Accordingly, cells treated with ON108110 rapidly accumulated in the G0/G1 stage of the cell cycle as a function of increasing concentration followed by rapid onset of apoptosis. Together, these results indicate that dual inhibition of CK2 and CDK4/6 may be an efficient treatment of MCL and T-ALLs displaying upregulation of CK2/PI3K and CDK4 signaling pathways.

7.
Nat Commun ; 8(1): 965, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042535

RESUMO

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N 2 amino group of guanine to generate four stereoisomeric BP-N 2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N 2-dG adducts, namely the 10S (+)-trans-BP-N 2-dG, 10R (+)-cis-BP-N 2-dG, and 10S ( - )-cis-BP-N 2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N 2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke that upon metabolic activation reacts with guanine. Here, the authors present the structures of the translesion DNA synthesis polymerase Rev1 in complex with three of the four possible stereoisomeric BP-N 2 -dG adducts, which gives insights how Rev1 achieves error-free replication.


Assuntos
Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Adutos de DNA/química , Reparo do DNA/fisiologia , Replicação do DNA , Guanina/química , Metais/química , Metais/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , Estereoisomerismo
8.
Sci Adv ; 2(10): e1601317, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27819052

RESUMO

PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP). The ability of PrimPol to function as a DNA primase stems from a simple but remarkable feature-almost complete lack of contacts to the DNA primer strand. This, in turn, allows two dNTPs to bind initiation and elongation sites on the enzyme for the formation of the first dinucleotide. PrimPol shows the ability to synthesize DNA opposite ultraviolet (UV) lesions; however, unexpectedly, the active-site cleft of the enzyme is constrained, which precludes the bypass of UV-induced DNA lesions by conventional translesion synthesis. Together, the structure addresses long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.


Assuntos
DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Enzimas Multifuncionais/química , DNA/biossíntese , DNA/genética , DNA Primase/genética , DNA Primase/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Raios Ultravioleta
9.
Nat Struct Mol Biol ; 19(6): 628-32, 2012 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-22562137

RESUMO

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Adutos de DNA/síntese química , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Domínio Catalítico , Cisplatino/síntese química , Cisplatino/química , Cisplatino/metabolismo , Cristalografia por Raios X , Adutos de DNA/química , Adutos de DNA/metabolismo , Nucleotídeos de Desoxicitosina/química , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Especificidade por Substrato
10.
Structure ; 19(6): 821-32, 2011 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-21645853

RESUMO

DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.


Assuntos
Adutos de DNA/química , DNA Polimerase beta/química , Reparo do DNA , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Mutagênese , Domínio Catalítico , Cristalografia por Raios X , Ensaios Enzimáticos , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/química
11.
Science ; 331(6020): 1036-40, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21163962

RESUMO

Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA/química , DNA/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Cisteína , DNA (Citosina-5-)-Metiltransferase 1 , DNA-Citosina Metilases/química , DNA-Citosina Metilases/metabolismo , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
12.
Nat Struct Mol Biol ; 17(3): 379-88, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20154704

RESUMO

The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3'-terminal cytosine opposite [AF]G in a nonrepetitive template sequence with low efficiency. This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex, with the 3'-terminal primer cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment. Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Fluorenos/química , Fluorenos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA Polimerase beta/genética , Cinética , Modelos Moleculares , Estrutura Secundária de Proteína , Difração de Raios X
13.
Structure ; 17(5): 725-36, 2009 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-19446528

RESUMO

7,8-Dihydro-8-oxoguanine (oxoG), the predominant oxidative DNA damage lesion, is processed differently by high-fidelity and Y-family lesion bypass polymerases. Although high-fidelity polymerases extend predominantly from an A base opposite an oxoG, the Y-family polymerases Dpo4 and human Pol eta preferentially extend from the oxoG*C base pair. We have determined crystal structures of extension Dpo4 ternary complexes with oxoG opposite C, A, G, or T and the next nascent base pair. We demonstrate that neither template backbone nor the architecture of the active site is perturbed by the oxoG(anti)*C and oxoG*A pairs. However, the latter manifest conformational heterogeneity, adopting both oxoG(syn)*A(anti) and oxoG(anti)*A(syn) alignment. Hence, the observed reduced primer extension from the dynamically flexible 3'-terminal primer base A is explained. Because of homology between Dpo4 and Pol eta, such a dynamic screening mechanism might be utilized by Dpo4 and Pol eta to regulate error-free versus error-prone bypass of oxoG and other lesions.


Assuntos
DNA Polimerase beta/química , Guanina/análogos & derivados , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/química , Dano ao DNA , DNA Polimerase beta/metabolismo , Reparo do DNA , Guanina/química , Guanina/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica
14.
Trends Biochem Sci ; 33(5): 209-19, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18407502

RESUMO

When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions.


Assuntos
Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Pareamento Incorreto de Bases , Benzo(a)pireno/farmacologia , DNA/efeitos dos fármacos , Dano ao DNA , DNA Polimerase beta/fisiologia , Guanina/análogos & derivados , Guanina/metabolismo , Modelos Moleculares , Transporte Proteico , Sulfolobus solfataricus/enzimologia
15.
Chem Res Toxicol ; 21(1): 45-52, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18052109

RESUMO

It has now been nearly two decades since the first solution structures of DNA duplexes covalently damaged by metabolically activated polycyclic aromatic hydrocarbons and amines were determined by NMR. Dozens of such high-resolution structures are now available, and some broad structural themes have been uncovered. It has been hypothesized that the solution structures are relevant to the biochemical processing of the adducts. The structural features of the adducts are considered to determine their mutational properties in DNA polymerases and their repair susceptibilities. In recent years, a number of crystal structures of DNA adducts of interest to our work have been determined in DNA polymerases. Accordingly, it is now timely to consider how NMR solution structures relate to structures within DNA polymerases. The NMR solution structural themes for polycyclic aromatic adducts are often observed in polymerase crystal structures. While the polymerase interactions can on occasion override the solution preferences, intrinsic adduct conformations favored in solution are often manifested within polymerases and likely play a significant role in lesion processing.


Assuntos
Adutos de DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Animais , DNA Polimerase Dirigida por DNA/química , Humanos , Espectroscopia de Ressonância Magnética , Mutação/genética , Conformação de Ácido Nucleico , Soluções , Relação Estrutura-Atividade
16.
Nucleic Acids Res ; 34(11): 3326-37, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16820532

RESUMO

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.


Assuntos
Adutos de DNA/química , DNA Polimerase beta/química , Desoxiguanosina/análogos & derivados , Desoxirribonucleotídeos/química , Imidazóis/química , Modelos Moleculares , Sítios de Ligação , Carcinógenos/química , Biologia Computacional , Simulação por Computador , DNA/química , DNA Polimerase beta/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Desoxiguanosina/química , Desoxirribonucleotídeos/metabolismo , Movimento (Física) , Mutagênese , Conformação de Ácido Nucleico , Sulfolobus solfataricus/enzimologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
17.
PLoS Biol ; 4(1): e11, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16379496

RESUMO

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG*C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.


Assuntos
Proteínas Arqueais/genética , DNA Polimerase beta/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas Arqueais/fisiologia , Sequência de Bases , Cristalização , Cristalografia por Raios X , DNA Polimerase beta/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Nucleotídeos de Desoxicitosina/metabolismo , Guanina/análogos & derivados , Guanina/fisiologia , Conformação de Ácido Nucleico , Conformação Proteica , Sulfolobus solfataricus/enzimologia , Moldes Genéticos , Translocação Genética
18.
Mutat Res ; 510(1-2): 23-35, 2002 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-12459440

RESUMO

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA/biossíntese , DNA/química , DNA/genética , Adutos de DNA/química , Adutos de DNA/genética , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estereoisomerismo
19.
J Biol Chem ; 277(46): 44582-7, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12228225

RESUMO

DNA polymerase mu (Polmu) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polmu function should be facilitated by an understanding of its biochemical activities. By using purified human Polmu for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N(6)-ethenoadenine, purified human Polmu efficiently bypassed the lesion. Even bulky DNA adducts such as N-2-acetylaminofluorene-adducted guanine, (+)- and (-)-trans-anti-benzo[a]pyrene-N(2)-dG were unable to block the polymerase activity of human Polmu. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polmu through a deletion mechanism. The Polmu specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polmu also effectively bypassed a template cis-syn TT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polmu and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases.


Assuntos
Adutos de DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Sequência de Bases , Sítios de Ligação , Dimerização , Humanos , Dados de Sequência Molecular , Mutação , Raios Ultravioleta
20.
J Biol Chem ; 277(34): 30488-94, 2002 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-12063247

RESUMO

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/fisiologia , Catálise , DNA Polimerase Dirigida por DNA/química , Humanos , Conformação de Ácido Nucleico , Estereoisomerismo
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