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1.
Mol Cell ; 76(1): 44-56.e3, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31444105

RESUMO

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.


Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Evolução Molecular , Inosina/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Animais , Proteínas Argonauta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Humanos , Magnésio/metabolismo , Manganês/metabolismo , Camundongos , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease H/genética , Relação Estrutura-Atividade , Especificidade por Substrato
2.
DNA Repair (Amst) ; 78: 45-59, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959406

RESUMO

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase that removes oxidized pyrimidines from DNA. The genome of Deinococcus radiodurans encodes for an unusually high number of DNA glycosylases, including three EndoIII enzymes (drEndoIII1-3). Here, we compare the properties of these enzymes to those of their well-studied homologues from E. coli and human. Our biochemical and mutational data, reinforced by MD simulations of EndoIII-DNA complexes, reveal that drEndoIII2 exhibits a broad substrate specificity and a catalytic efficiency surpassing that of its counterparts. In contrast, drEndoIII1 has much weaker and uncoupled DNA glycosylase and AP-lyase activities, a characteristic feature of eukaryotic DNA glycosylases, and was found to present a relatively robust activity on single-stranded DNA substrates. To our knowledge, this is the first report of such an activity for an EndoIII. In the case of drEndoIII3, no catalytic activity could be detected, but its ability to specifically recognize lesion-containing DNA using a largely rearranged substrate binding pocket suggests that it may play an alternative role in genome maintenance. Overall, these findings reveal that D. radiodurans possesses a unique set of DNA repair enzymes, including three non-redundant EndoIII variants with distinct properties and complementary activities, which together contribute to genome maintenance in this bacterium.


Assuntos
Reparo do DNA , DNA Complementar/genética , Deinococcus/enzimologia , Deinococcus/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Mutação , Biocatálise , DNA Complementar/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Pirimidinas/metabolismo , Especificidade por Substrato
3.
Fam Cancer ; 18(2): 179-182, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30859360

RESUMO

While familial adenomatous polyposis accounts for approximately 1% of all colorectal cancer, the genetic cause underlying the development of multiple colonic adenomas remains unsolved in many patients. Adenomatous polyposis syndromes can be divided into: familial adenomatous polyposis, MUTYH-associated polyposis, polymerase proofreading associated polyposis and the recently described NTHL1-associated polyposis (NAP). NAP is characterised by recessive inheritance, attenuated adenomatous polyposis, colonic cancer(s) and possible extracolonic malignancies. To date, 11 cases have been reported as having germline homozygous or compound heterozygous mutations in the base excision repair gene NTHL1. Here we present a further case of a 65-year-old male with a history of adenomatous polyposis and bladder cancer, who has a previously described homozygous nonsense variant in the NTHL1 gene. This case is consistent with the emerging phenotype previously described of multiple colorectal adenomas and at least one primary tumour, adding to the small but growing body of literature about NAP.


Assuntos
Polipose Adenomatosa do Colo/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Neoplasias da Bexiga Urinária/genética , Polipose Adenomatosa do Colo/diagnóstico , Idoso , Austrália , Mutação em Linhagem Germinativa , Humanos , Masculino , Neoplasias da Bexiga Urinária/diagnóstico
4.
Adv Genet ; 103: 183-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30904095

RESUMO

Colorectal carcinoma (CRC) is the third most common cancer in men and the second most common cancer in women across the world. Most CRCs occur sporadically, but in 15-35% of cases, hereditary factors are important. Some patients with an inherited predisposition to CRC will be diagnosed with a "genetic polyposis syndrome" such as familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), polymerase proofreading associated polyposis (PPAP), NTHL1-associated polyposis, MSH3-associated polyposis or a hamartomatous polyposis syndrome. Individuals with ≥10 colorectal polyps have traditionally been referred for genetic diagnostic testing to identify APC and MUTYH mutations which cause FAP and MAP respectively. Mutations are found in most patients with >100 adenomas but in only a minority of those with 10-100 adenomas. The reasons that diagnostic laboratories are not identifying pathogenic variants include mutations occurring outside of the open reading frames of genes, individuals exhibiting generalized mosaicism and the involvement of additional genes. It is important to identify patients with an inherited polyposis syndrome, and to define the mutations causing their polyposis, so that the individuals and their relatives can be managed appropriately.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Variação Genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Mutação , Fenótipo
5.
PLoS One ; 14(2): e0211653, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30726292

RESUMO

DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B. subtilis YwqL, a homolog of Endonuclease-V (EndoV), acts on a wider spectrum of deaminated bases but the factors that complete this pathway have remained elusive. Here, we report that a purified His6-YwqL (hereafter BsEndoV) protein had in vitro endonuclease activity against double-stranded DNAs containing a single uracil (U), hypoxanthine (Hx), xanthine (X) or an AP site. Interestingly, while BsEndoV catalyzed a single strand break at the second phosphodiester bond towards the 3'-end of the U and AP lesions, there was an additional cleavage of the phosphodiester bond preceding the Hx and X lesions. Remarkably, the repair event initiated by BsEndoV on Hx and X, was completed by a recombinant B. subtilis His6-DNA polymerase A (BsPolA), but not on BsEndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B. subtilis His6-ExoA (BsExoA) was necessary before completion of their repair by BsPolA. These results suggest the existence of a novel alternative excision repair pathway in B. subtilis that counteracts the genotoxic effects of base deamination. The presence of this novel pathway in vivo in B. subtilis was also supported by analysis of effects of single or multiple deletions of exoA, endoV and polA on spontaneous mutations in growing cells, and the sensitivity of growing wild-type and mutant cells to a DNA deaminating agent.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Polimerase I/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Polimerase I/genética , Desaminação , Desoxirribonuclease (Dímero de Pirimidina)/genética , Mutagênese , Proteínas Recombinantes
6.
Cancer Cell ; 35(2): 256-266.e5, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753826

RESUMO

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.


Assuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Perfilação da Expressão Gênica , Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias/genética , Transcriptoma , Adulto , Idoso , Biomarcadores Tumorais/deficiência , Reparo do DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/deficiência , Europa (Continente) , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Neoplásicas Hereditárias/enzimologia , Síndromes Neoplásicas Hereditárias/patologia , Linhagem , Fenótipo , Medição de Risco , Fatores de Risco , Adulto Jovem
7.
Nucleic Acids Res ; 47(6): 2922-2931, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30649547

RESUMO

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be 'handed' from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3' diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Nucleossomos/enzimologia , Cromatina/enzimologia , Cromatina/genética , DNA/química , Dano ao DNA/genética , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Esterases/genética , Humanos , Liases/química , Liases/genética , Nucleossomos/genética , Oxirredução
8.
Free Radic Biol Med ; 131: 264-273, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552997

RESUMO

The NTHL1 gene encodes DNA glycosylase, which is involved in base excision repair, and biallelic mutations of this gene result in NTHL1-associated polyposis (NAP), a hereditary disease characterized by colorectal polyposis and multiple types of carcinomas. However, no proper functional characterization of variant NTHL1 proteins has been done so far. Herein, we report functional evaluation of variant NTHL1 proteins to aid in the accurate diagnosis of NAP. First, we investigated whether it would be appropriate to use 5-hydroxyuracil (5OHU), an oxidation product of cytosine, for the evaluation. In the supF forward mutation assay, 5OHU caused an increase of the mutation frequency in human cells, and the C→T mutation was predominant among the 5OHU-induced mutations. In addition, in DNA cleavage activity assay, 5OHU was excised by NTHL1 as well as four other DNA glycosylases (SMUG1, NEIL1, TDG, and UNG2). When human cells overexpressing the five DNA glycosylases were established, it was found that each of the five DNA glycosylases, including NTHL1, had the ability to suppress 5OHU-induced mutations. Based on the above results, we performed functional evaluation of eight NTHL1 variants using 5OHU-containing DNA substrate or shuttle plasmid. The DNA cleavage activity assay showed that the variants of NTHL1, Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, showed defective repair activity for 5OHU and two other oxidatively damaged bases. Moreover, the supF forward mutation assay showed that the four truncated-type NTHL1 variants showed a reduced ability to suppress 5OHU-induced mutations in human cells. These results suggest that the NTHL1 variants Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, were defective in 5OHU repair and the alleles encoding them were considered to be pathogenic for NAP.


Assuntos
Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Uracila/análogos & derivados , Polipose Adenomatosa do Colo/diagnóstico , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Alelos , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Clivagem do DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Análise Mutacional de DNA , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Humanos , Mutação , Uracila/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
10.
J Biosci ; 43(4): 575-583, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30207305

RESUMO

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.


Assuntos
Dano ao DNA/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Extratos Celulares/genética , Extratos Celulares/farmacologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos da radiação , DNA Glicosilases/farmacologia , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Desoxiguanosina/análogos & derivados , Desoxiguanosina/genética , Desoxirribonuclease (Dímero de Pirimidina)/farmacologia , Células HeLa , Humanos , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Radiação Ionizante , Timina/análogos & derivados
11.
Hum Genet ; 137(10): 795-806, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30267214

RESUMO

Although ~ 25% of colorectal cancer or polyp (CRC/P) cases show familial aggregation, current germline genetic testing identifies a causal genotype in the 16 major genes associated with high penetrance CRC/P in only 20% of these cases. As there are likely other genes underlying heritable CRC/P, we evaluated the association of variation at novel loci with CRC/P. We evaluated 158 a priori selected candidate genes by comparing the number of rare potentially disruptive variants (PDVs) found in 84 CRC/P cases without an identified CRC/P risk-associated variant and 2440 controls. We repeated this analysis using an additional 73 CRC/P cases. We also compared the frequency of PDVs in select genes among CRC/P cases with two publicly available data sets. We found a significant enrichment of PDVs in cases vs. controls: 20% of cases vs. 11.5% of controls with ≥ 1 PDV (OR = 1.9, p = 0.01) in the original set of cases. Among the second cohort of CRC/P cases, 18% had a PDV, significantly different from 11.5% (p = 0.02). Logistic regression, adjusting for ancestry and multiple testing, indicated association between CRC/P and PDVs in NTHL1 (p = 0.0001), BRCA2 (p = 0.01) and BRIP1 (p = 0.04). However, there was no significant difference in the frequency of PDVs at each of these genes between all 157 CRC/P cases and two publicly available data sets. These results suggest an increased presence of PDVs in CRC/P cases and support further investigation of the association of NTHL1, BRCA2 and BRIP1 variation with CRC/P.


Assuntos
Proteína BRCA2/genética , Neoplasias Colorretais/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Loci Gênicos , Variação Genética , RNA Helicases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
12.
Int J Biol Macromol ; 117: 17-24, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29800668

RESUMO

Endonuclease V (Endo V) is an important enzyme for repairing deoxyinosine in DNA. While bacterial and eukaryotic endo Vs have been well studied, knowledge of archaeal endo Vs is limited. Here, we first presented biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba endo V). The recombinant enzyme possessed optimal endonuclease activity for cleaving deoxyinosine-containing DNA at 70-90 °C. Furthermore, Tba endo V can withstand 100 °C for 120 min without significant loss of its activity, suggesting the enzyme is thermostable. Tba endo V exhibited varying cleavage efficiencies at various pH levels from 6.0 to 11.0, among which an optimal pH for the enzyme was 8.0-9.0. In addition, a divalent metal ion was required for the enzyme to cleave DNA. Mn2+ and Mg2+ were optimal ions for the enzyme's activity whereas Ca2+, Zn2+ and Co2+ inhibited the enzyme activity. Moreover, the enzyme activity was suppressed by high NaCl concentration. Tba endo V bound to all DNA substrates; however, the enzyme exhibited a higher affinity for binding to deoxyinosine-containing DNA than normal DNA. Our work provides valuable information for revealing the role of Tba endo V in the base excision repair pathway for deoxyinosine repair in Thermococcus.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Thermococcus/enzimologia , Sequência de Aminoácidos , Clivagem do DNA , Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/isolamento & purificação , Ativação Enzimática , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Íons/química , Cinética , Metais/química , Proteínas Recombinantes , Especificidade por Substrato , Temperatura Ambiente , Thermococcus/genética
13.
Artigo em Inglês | MEDLINE | ID: mdl-29685966

RESUMO

Cytosine (C) in DNA is often modified to 5-methylcytosine (m5C) to execute important cellular functions. Despite the significance of m5C for epigenetic regulation in mammals, damage to m5C has received little attention. For instance, almost no studies exist on erroneous methylation of m5C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m5C into N4,5-dimethylcytosine (m N4,5C) in DNA, m N4,5C is probably present in vivo We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N4,5C residues in vitro The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m5C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N4,5C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.


Assuntos
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , DNA-Formamidopirimidina Glicosilase/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Epigênese Genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Citosina/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Metilação
14.
Nucleic Acids Res ; 46(9): 4515-4532, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29522130

RESUMO

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.


Assuntos
Transformação Celular Neoplásica , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Instabilidade Genômica , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Dano ao DNA , Replicação do DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Células Epiteliais/enzimologia , Humanos , Neoplasias Pulmonares/enzimologia , Mutação , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia
15.
J Pathol ; 244(2): 135-142, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29105096

RESUMO

It is now well established that germline genomic aberrations can underlie high-penetrant familial polyposis and colorectal cancer syndromes, but a genetic cause has not yet been found for the major proportion of patients with polyposis. Since next-generation sequencing has become widely accessible, several novel, but rare, high-penetrant risk factors for adenomatous polyposis have been identified, all operating in pathways responsible for genomic maintenance and DNA repair. One of these is the base excision repair pathway. In addition to the well-established role of the DNA glycosylase gene MUTYH, biallelic mutations in which predispose to MUTYH-associated polyposis, a second DNA glycosylase gene, NTHL1, has recently been associated with adenomatous polyposis and a high colorectal cancer risk. Both recessive polyposis syndromes are associated with increased risks for several other cancer types as well, but the spectrum of benign and malignant tumours in individuals with biallelic NTHL1 mutations was shown to be broader; hence the name NTHL1-associated tumour syndrome. Colorectal tumours encountered in patients with these syndromes show unique, clearly distinct mutational signatures that may facilitate the identification of these syndromes. On the basis of the prevalence of pathogenic MUTYH and NTHL1 variants in the normal population, we estimate that the frequency of the novel NTHL1-associated tumour syndrome is five times lower than that of MUTYH-associated polyposis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Polipose Adenomatosa do Colo/genética , Biomarcadores Tumorais/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Mutação , Polipose Adenomatosa do Colo/epidemiologia , Polipose Adenomatosa do Colo/patologia , Animais , Dano ao DNA , Predisposição Genética para Doença , Humanos , Taxa de Mutação , Penetrância , Fenótipo , Fatores de Risco
16.
Science ; 358(6360): 234-238, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28912133

RESUMO

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations.


Assuntos
Sistemas CRISPR-Cas , Colo , Desoxirribonuclease (Dímero de Pirimidina)/genética , Proteína 1 Homóloga a MutL/genética , Neoplasias/genética , Organoides , Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/genética , Replicação do DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Mutação INDEL , Mutagênese , Células-Tronco
17.
Sci Rep ; 7(1): 8505, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819113

RESUMO

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.


Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismo , Grânulos Citoplasmáticos/enzimologia , Citosol/enzimologia , DNA de Protozoário/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/deficiência , Desoxirribonuclease (Dímero de Pirimidina)/genética , Técnicas de Silenciamento de Genes , Especificidade por Substrato , Trypanosoma brucei brucei/genética
18.
Sci Rep ; 7(1): 2214, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28533537

RESUMO

Colo-Rectal Cancer is a common cancer worldwide with 5-10% cases being hereditary. Familial Adenomatous Polyposis (FAP) syndrome is due to germline mutations in the APC or rarely MUTYH gene. NTHL1, POLD1, POLE have been recently reported in previously unexplained FAP cases. Unlike the Caucasian population, FAP phenotype and its genotypic associations have not been widely studied in several geoethnic groups. We report the first FAP cohort from South Asia and the only non-Caucasian cohort with comprehensive analysis of APC, MUTYH, NTHL1, POLD1, POLE genes. In this cohort of 112 individuals from 53 FAP families, we detected germline APC mutations in 60 individuals (45 families) and biallelic MUTYH mutations in 4 individuals (2 families). No NTHL1, POLD1, POLE mutations were identified. Fifteen novel APC mutations and a new Indian APC mutational hotspot at codon 935 were identified. Eight very rare FAP phenotype or phenotypes rarely associated with mutations outside specific APC regions were observed. APC genotype-phenotype association studies in different geo-ethnic groups can enrich the existing knowledge about phenotypic consequences of distinct APC mutations and guide counseling and risk management in different populations. A stepwise cost-effective mutation screening approach is proposed for genetic testing of south Asian FAP patients.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , DNA Glicosilases/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Polipose Adenomatosa do Colo/diagnóstico , Alelos , Éxons , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Índia , Masculino , Mutação , Fenótipo
19.
DNA Repair (Amst) ; 53: 43-51, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28292631

RESUMO

The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.


Assuntos
Domínio Catalítico , Dano ao DNA , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Mutação , Sequência de Aminoácidos , DNA/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Humanos , Cinética , Especificidade por Substrato
20.
Proc Natl Acad Sci U S A ; 114(10): 2604-2609, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28143930

RESUMO

Reactive oxygen species (ROS) have emerged as important cellular-signaling agents for cellular survival. Herein, we demonstrate that ROS-mediated oxidation of DNA to yield 8-oxo-7,8-dihydroguanine (OG) in gene promoters is a signaling agent for gene activation. Enhanced gene expression occurs when OG is formed in guanine-rich, potential G-quadruplex-forming sequences (PQS) in promoter-coding strands, initiating base excision repair (BER) by 8-oxoguanine DNA glycosylase (OGG1), yielding an abasic site (AP). The AP enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold in which apurinic/apyrimidinic endonuclease 1 (APE1) binds, but inefficiently cleaves, the AP for activation of vascular endothelial growth factor (VEGF) or endonuclease III-like protein 1 (NTHL1) genes. These details were mapped via synthesis of OG and AP analogs at single-nucleotide precision within the promoter of a luciferase reporter system. The reporters were analyzed in human and mouse cells while selectively knocking out or down critical BER proteins to identify the impact on luciferase expression. Identification of the oxidatively modified DNA base OG to guide BER activity in a gene promoter and impact cellular phenotype ascribes an epigenetic role to OG.


Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Estresse Oxidativo/genética , Animais , DNA Glicosilases/química , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Epigênese Genética/genética , Quadruplex G , Regulação da Expressão Gênica/genética , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Transcrição Genética/genética , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética
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