RESUMO
RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.
Assuntos
DNA Helicases , Humanos , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/genética , Cristalografia por Raios X , Modelos Moleculares , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Domínios Proteicos , Espalhamento a Baixo ÂnguloRESUMO
Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.
Assuntos
Proteína BRCA2 , Neoplasias da Mama , Glicólise , Aldeído Pirúvico , Animais , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Camundongos , Humanos , Feminino , Aldeído Pirúvico/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Haploinsuficiência , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Mutação , Dano ao DNA , Reparo do DNA , Linhagem Celular TumoralRESUMO
Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague-Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.
RESUMO
Sus1p is a common component of transcriptional co-activator, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and mRNA export complex, TREX-2 (Transcription-export 2), and is involved in promoting transcription and mRNA export. However, it is not clearly understood how Sus1p promotes transcription. Here, we show that Sus1p is predominantly recruited to the upstream activating sequence of a SAGA-dependent gene, GAL1, under transcriptionally active conditions as a component of SAGA to promote the formation of pre-initiation complex (PIC) at the core promoter and, consequently, transcriptional initiation. Likewise, Sus1p promotes the PIC formation at other SAGA-dependent genes and hence transcriptional initiation. Such function of Sus1p in promoting PIC formation and transcriptional initiation is not mediated via its role in regulation of SAGA's histone H2B de-ubiquitylation activity. However, Sus1p's function in regulation of histone H2B ubiquitylation is associated with transcriptional elongation, DNA repair and replication. Collectively, our results support that Sus1p promotes PIC formation (and hence transcriptional initiation) at the SAGA-regulated genes independently of histone H2B de-ubiquitylation and further controls transcriptional elongation, DNA repair and replication via orchestration of histone H2B ubiquitylation, thus providing distinct functional insights of Sus1p in regulation of DNA transacting processes.
Assuntos
Replicação do DNA , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação da Cromatina , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Galactoquinase , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transcrição Gênica , Ativação Transcricional , UbiquitinaçãoRESUMO
Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB.