RESUMEN
A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Inestabilidad Genómica , Células HeLa , Humanos , Cinética , Modelos Genéticos , Ftalazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
The process of DNA replication includes duplex unwinding, followed immediately by DNA synthesis. In eukaryotes, DNA synthesis is disturbed in damaged DNA regions, in replication slow zones, or as a result of insufficient nucleotide level. This review aims to discuss the mechanisms that coordinate DNA unwinding and synthesis, allowing replication to be completed even in the presence of genomic insults. There is a growing body of evidence which suggests that S-phase checkpoint pathways regulate both replicative unwinding and DNA synthesis, to synchronize the two processes, thus ensuring genome stability.
Asunto(s)
Replicación del ADN/fisiología , Fase S/fisiología , Saccharomyces cerevisiae/genética , Animales , ADN/biosíntesis , ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae/metabolismo , XenopusRESUMEN
The essential cis- and trans-acting elements required for RNA splicing have been defined, however, the detailed molecular mechanisms underlying intron-exon recognition are still unclear. Here we demonstrate that the ratio between stability of mRNA/DNA and DNA/DNA duplexes near 3'-spice sites is a characteristic feature that can contribute to intron-exon differentiation. Remarkably, throughout all transcripts, the most unstable mRNA/DNA duplexes, compared with the corresponding DNA/DNA duplexes, are situated upstream of the 3'-splice sites and include the polypyrimidine tracts. This characteristic instability is less pronounced in weak alternative splice sites and disease-associated cryptic 3'-splice sites. Our results suggest that this thermodynamic pattern can prevent the re-annealing of mRNA to the DNA template behind the RNA polymerase to ensure access of the splicing machinery to the polypyrimidine tract and the branch point. In support of this mechanism, we demonstrate that RNA/DNA duplex formation at this region prevents pre-spliceosome A complex assembly.
Asunto(s)
Eucariontes/genética , Exones/genética , Intrones/genética , Empalme Alternativo/genética , Animales , Emparejamiento Base/genética , Secuencia de Bases , Caenorhabditis elegans/genética , ADN/metabolismo , Genoma de los Helmintos/genética , Humanos , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , Nucleótidos/genética , Sitios de Empalme de ARN , Estabilidad del ARN/genética , Empalmosomas/metabolismo , TermodinámicaRESUMEN
Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3'-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3'-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription.