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
Cells are constantly exposed to numerous mutagens that produce diverse types of DNA lesions. Eukaryotic cells have evolved an impressive array of DNA repair mechanisms that are able to detect and repair these lesions, thus preventing genomic instability. The DNA repair process is subjected to precise spatiotemporal coordination, and repair proteins are recruited to lesions in an orderly fashion, depending on their function. Here, we present DNArepairK, a unique open-access database that contains the kinetics of recruitment and removal of 70 fluorescently tagged DNA repair proteins to complex DNA damage sites in living HeLa Kyoto cells. An interactive graphical representation of the data complemented with live cell imaging movies facilitates straightforward comparisons between the dynamics of proteins contributing to different DNA repair pathways. Notably, most of the proteins included in DNArepairK are represented by their kinetics in both nontreated and PARP1/2 inhibitor-treated (talazoparib) cells, thereby providing an unprecedented overview of the effects of anticancer drugs on the regular dynamics of the DNA damage response. We believe that the exclusive dataset available in DNArepairK will be of value to scientists exploring the DNA damage response but, also, to inform and guide the development and evaluation of novel DNA repair-targeting anticancer drugs.