Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.138
Filtrar
1.
Top Curr Chem (Cham) ; 378(2): 32, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32146604

RESUMO

DNA hydrogels are crosslinked polymeric networks in which DNA is used as the backbone or the crosslinker. These hydrogels are novel biofunctional materials that possess the biological character of DNA and the framed structure of hydrogels. Compared with other kinds of hydrogels, DNA hydrogels exhibit not only high mechanical strength and controllable morphologies but also good recognition ability, designable responsiveness, and programmability. The DNA used in this type of hydrogel acts as a building block for self-assembly or as a responsive element due to its sequence recognition ability and switchable structural transitions, respectively. In this review, we describe recent developments in the field of DNA hydrogels and discuss the role played by DNA in these hydrogels. Various synthetic strategies for and a range of applications of DNA hydrogels are detailed.


Assuntos
DNA/química , Hidrogéis/química , DNA Polimerase Dirigida por DNA/metabolismo , Ligases/metabolismo , Luz , Nanotecnologia , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico
2.
Chem Commun (Camb) ; 56(14): 2186-2189, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-31971182

RESUMO

The intrinsic l-DNA binding properties of a natural DNA polymerase was discovered. The binding affinity of Dpo4 polymerase for l-DNA was comparable to that for d-DNA. The crystal structure of Dpo4/l-DNA complex revealed a dimer formed by the little finger domain that provides a binding site for l-DNA.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/química , Cristalografia por Raios X , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica
3.
J Chem Theory Comput ; 16(2): 1319-1332, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31972079

RESUMO

Proteins in vivo endure highly various interactions from the luxuriant surrounding macromolecular cosolutes. Confinement and macromolecular crowding are the two major effects that should be considered while comparing the results of protein dynamics from in vitro to in vivo. However, efforts have been largely focused on single domain protein folding up to now, and the quantifications of the in vivo effects in terms of confinements and crowders on modulating the structure and dynamics as well as the physical understanding of the underlying mechanisms on multidomain protein folding are still challenging. Here we developed a topology-based model to investigate folding of a multidomain Y-family DNA polymerase (DPO4) within spherical confined space and in the presence of repulsive and attractive crowders. We uncovered that the entropic component of the thermodynamic driving force led by confinements and repulsive crowders increases the stability of folded states relative to the folding intermediates and unfolded states, while the enthalpic component of the thermodynamic driving force led by attractive crowders gives rise to the opposite effects with less stability. We found that the shapes of DPO4 conformations influenced by the confinements and the crowders are quite different even when only the entropic component of the thermodynamic driving force is considered. We uncovered that under all in vivo conditions, the folding cooperativity of DPO4 decreases compared to that in bulk. We showed that the loss of folding cooperativity can promote the sequential domain-wise folding, which was widely found in cotranslational multidomain protein folding, and effectively prohibit the backtracking led by topological frustrations during multidomain protein folding processes.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Dobramento de Proteína , Simulação por Computador , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Termodinâmica
4.
Cell Mol Life Sci ; 77(1): 35-59, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31722068

RESUMO

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Domínio Catalítico , Reparo do DNA , Replicação do DNA , Humanos , Modelos Moleculares , Conformação Proteica
5.
Chemistry ; 26(6): 1286-1291, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31725178

RESUMO

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.


Assuntos
DNA/química , Compostos Ferrosos/química , Metalocenos/química , Nucleotídeos/química , Coloração e Rotulagem/métodos , Citidina Trifosfato/química , DNA/metabolismo , Sondas de DNA/síntese química , Sondas de DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas Eletroquímicas , Oxirredução , Especificidade por Substrato
6.
Chem Pharm Bull (Tokyo) ; 67(10): 1123-1130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582632

RESUMO

The adenosine triphosphate derivatives of 2-oxo-1,3-diazaphenoxazine (dAdapTP) showed a significant discrimination ability for the template strand including that between 8-oxo-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) by the single nucleotide primer extension reaction using the Klenow Fragment. In this study, we synthesized new dAdapTP derivatives, i.e., 2-amino-dAdapTP, 2-chloro-dAdapTP and 2-iodo-dAdapTP, to investigate the effect on the selectivity and efficiency of incorporation for the primer extension reaction using a variety of DNA polymerases. In contrast to the previously tested dAdapTP, the selectivity and efficiency of the 2-halo-dAdapTP incorporation were dramatically decreased using the Klenow Fragment. Moreover, the efficiency of the 2-amino-dAdapTP incorporation into the T-containing template was almost the same with that of dAdapTP. In the case of the Bsu DNA polymerase, the efficiency of all the dAdapTP derivatives decreased compared to that using the Klenow Fragment. However, the incorporation selectivity of dAdapTP had improved against the oxodG-containing template for all the template sequences including the T-containing template. Moreover, 2-amino-dAdapTP showed a better efficiency than dAdapTP using the Bsu DNA polymerase. The 2-amino group of the adenosine unit may interact with syn-oxodG at the active site of the Bsu DNA polymerase during the single primer extension reaction.


Assuntos
Adenosina/metabolismo , Compostos Aza/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Oxazinas/metabolismo , Polifosfatos/metabolismo , Adenosina/química , Compostos Aza/química , DNA Polimerase Dirigida por DNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Estrutura Molecular , Oxazinas/química , Polifosfatos/química
7.
Nat Struct Mol Biol ; 26(10): 955-962, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31582849

RESUMO

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.


Assuntos
DNA Polimerase III/química , DNA Polimerase Dirigida por DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Microscopia Crioeletrônica , DNA Polimerase III/metabolismo , DNA Polimerase III/ultraestrutura , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
8.
Postepy Biochem ; 65(2): 143-152, 2019 06 06.
Artigo em Polonês | MEDLINE | ID: mdl-31642653

RESUMO

High replication fidelity, understood as the DNA polymerases' ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme's catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , DNA/metabolismo , Instabilidade Genômica , Ribonucleotídeos/metabolismo , DNA/genética , Replicação do DNA , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Ribonucleotídeos/química
9.
Enzymes ; 45: 139-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627876

RESUMO

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death. To avoid this, cells possess additional DNA polymerases such as the Y-family of polymerases and the B-family member Polζ that can replicate over sites of DNA damage in a process called translesion synthesis (TLS). While able to replicate over DNA lesions, the TLS polymerases exhibit low-fidelity on undamaged DNA and, consequently, must be prevented from replicating DNA under normal circumstances and recruited only when necessary. The replicative bypass of most types of DNA lesions requires the consecutive action of these specialized TLS polymerases assembled into a dynamic multiprotein complex called the Rev1/Polζ mutasome. To this end, posttranslational modifications and a network of protein-protein interactions mediated by accessory domains/subunits of the TLS polymerases control the assembly and rearrangements of the Rev1/Polζ mutasome and recruitment of TLS proteins to sites of DNA damage. This chapter focuses on the structures and interactions that control these processes underlying the function of the Rev1/Polζ mutasome, as well as the development of small molecule inhibitors of the Rev1/Polζ-dependent TLS holding promise as a potential anticancer therapy.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética
10.
Enzymes ; 45: 183-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627877

RESUMO

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Complexos Multienzimáticos/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/química
11.
Enzymes ; 45: 225-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627878

RESUMO

Despite their relative simplicity, iron-sulfur clusters have been omnipresent as cofactors in myriad cellular processes such as oxidative phosphorylation and other respiratory pathways. Recent research advances confirm the presence of different clusters in enzymes involved in nucleic acid metabolism. Iron-sulfur clusters can therefore be considered hallmarks of cellular metabolism. Helicases, nucleases, glycosylases, DNA polymerases and transcription factors, among others, incorporate various types of clusters that serve differing roles. In this chapter, we review our current understanding of the identity and functions of iron-sulfur clusters in DNA and RNA metabolizing enzymes, highlighting their importance as regulators of cellular function.


Assuntos
Coenzimas/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Ácidos Nucleicos/metabolismo , Coenzimas/química , DNA Glicosilases/química , DNA Glicosilases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo
12.
Enzymes ; 45: 289-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627881

RESUMO

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.


Assuntos
DNA Primase/metabolismo , Primers do DNA/biossíntese , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Humanos
13.
PLoS Pathog ; 15(10): e1008111, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626661

RESUMO

The herpes simplex virus virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs and blunts host innate antiviral responses. Previous work demonstrated that cells infected with vhs mutants display enhanced activation of the host double-stranded RNA (dsRNA)-activated protein kinase R (PKR), implying that vhs limits dsRNA accumulation in infected cells. Confirming this hypothesis, we show that partially complementary transcripts of the UL23/UL24 and UL30/31 regions of the viral genome increase in abundance when vhs is inactivated, giving rise to greatly increased levels of intracellular dsRNA formed by annealing of the overlapping portions of these RNAs. Thus, vhs limits accumulation of dsRNA at least in part by reducing the levels of complementary viral transcripts. We then asked if vhs also destabilizes dsRNA after its initial formation. Here, we used a reporter system employing two mCherry expression plasmids bearing complementary 3' UTRs to produce defined dsRNA species in uninfected cells. The dsRNAs are unstable, but are markedly stabilized by co-expressing the HSV dsRNA-binding protein US11. Strikingly, vhs delivered by super-infecting HSV virions accelerates the decay of these pre-formed dsRNAs in both the presence and absence of US11, a novel and unanticipated activity of vhs. Vhs binds the host RNA helicase eIF4A, and we find that vhs-induced dsRNA decay is attenuated by the eIF4A inhibitor hippuristanol, providing evidence that eIF4A participates in the process. Our results show that a herpesvirus host shutoff RNase destabilizes dsRNA in addition to targeting partially complementary viral mRNAs, raising the possibility that the mRNA destabilizing proteins of other viral pathogens dampen the host response to dsRNA through similar mechanisms.


Assuntos
Estabilidade de RNA/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribonucleases/metabolismo , Simplexvirus/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/metabolismo , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Vero
14.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569643

RESUMO

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA/química , Guanina/análogos & derivados , Mutagênicos/farmacologia , DNA Polimerase beta/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/química , Guanina/farmacologia , Halogenação , Humanos , Ligações de Hidrogênio , Cinética , Modelos Biológicos , Conformação Molecular , Mutagênicos/química
15.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569730

RESUMO

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10-15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Plantas/enzimologia , Plantas/genética , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , Epigenômica/métodos , Genoma de Planta , Meiose , Subunidades Proteicas , Origem de Replicação , Transdução de Sinais , Estresse Fisiológico
16.
Nat Commun ; 10(1): 4423, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562312

RESUMO

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Domínio Catalítico , Quebras de DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida
17.
Nat Commun ; 10(1): 4286, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537809

RESUMO

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.


Assuntos
Neoplasias da Mama/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Aminoquinolinas/toxicidade , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/metabolismo , Células HEK293 , Humanos , Camundongos , Mitomicina/toxicidade , Ácidos Picolínicos/toxicidade
18.
Int J Radiat Biol ; 95(12): 1613-1626, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31498026

RESUMO

Purpose: The aim of this paper was to investigate the sequence preference of ionizing radiation (IR)-induced DNA damage as assessed by a linear amplification/polymerase stop (LA/PS) assay. The LA/PS assay is able to detect a wide range of IR-induced DNA lesions and this technique was utilized to quantitatively determine the preferential sites of gamma irradiation-induced DNA lesions in three different DNA sequences.Materials and methods: This analysis was performed on an automated DNA sequencer with capillary electrophoresis and laser-induced fluorescence detection.Results: The main outcome of this study was that G nucleotides were preferentially found at IR-induced polymerase stop sites. The individual nucleotides at the IR-induced DNA damage sites were analyzed and a consensus sequence of 5'-GG* (where * indicates the damaged nucleotide) was observed. In a separate method of analysis, the dinucleotides and trinucleotides at the IR-induced DNA damage sites were examined and 5'-GG* and 5'-G*G dinucleotides and 5'-GG*G trinucleotides were found to be the most prevalent. The use of the LA/PS assay permits a large number of IR-induced DNA lesions to be detected in the one procedure including: double- and single-strand breaks, apurinic/apyrimidinic sites and base damage.Conclusions: It was concluded that 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G) and the degradation products of 8-oxoG were possibly the main lesions detected. To our knowledge, this is the first occasion that the DNA sequence preference of IR-induced DNA damage as detected by a LA/PS assay has been reported.


Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Raios gama/efeitos adversos , Sequência de Bases , Oligonucleotídeos/genética , Plasmídeos/genética
20.
PLoS Genet ; 15(8): e1008319, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381562

RESUMO

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12-20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Mutação , Neoplasias/tratamento farmacológico , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Mutações Sintéticas Letais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA