RESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Colo do Útero/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Instabilidade Genômica , Células HeLa , Humanos , Cinética , Modelos Genéticos , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
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.
Assuntos
Eucariotos/genética , Éxons/genética , Íntrons/genética , Processamento Alternativo/genética , Animais , Pareamento de Bases/genética , Sequência de Bases , Caenorhabditis elegans/genética , DNA/metabolismo , Genoma Helmíntico/genética , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/metabolismo , Nucleotídeos/genética , Sítios de Splice de RNA , Estabilidade de RNA/genética , Spliceossomos/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
DNA/química , DNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Saccharomyces/genética , Transcrição Gênica , Cromossomos Fúngicos/genética , DNA Fúngico/genética , Estabilidade de Medicamentos , Íntrons/genética , Fases de Leitura Aberta , RNA Fúngico/genética , TermodinâmicaRESUMO
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.