RESUMO
During DNA repair, ATM-induced H2AX histone phosphorylation and MDC1 recruitment spread megabases beyond the damage site. While loop extrusion has been suggested to drive this spread, the underlying mechanism remains unclear. Herein, we provide two lines of evidence that loop extrusion is not the only driver of damage-induced γH2AX spread. First, cohesin loader NIPBL and cohesin subunit RAD21 accumulate considerably later than the phosphorylation of H2AX and MDC1 recruitment at micro-IR-induced damage. Second, auxin-induced RAD21 depletion does not affect γH2AX/MDC1 spread following micro-irradiation or DSB induction by zeocin. To determine if diffusion of activated ATM could account for the observed behavior, we measured the exchange rate and diffusion constants of ATM and MDC1 within damaged and unperturbed chromatin. Using these measurements, we introduced a quantitative model in which the freely diffusing activated ATM phosphorylates H2AX. This model faithfully describes the dynamics of ATM and subsequent γH2AX/MDC1 spread at complex DNA lesions.
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.