RESUMEN
Accurate replication of the genome is fundamental to cellular survival and tumor prevention. The DNA replication fork is vulnerable to DNA lesions and damages that impair replisome progression, and improper control over DNA replication stress inevitably causes fork stalling and collapse, a major source of genome instability that fuels tumorigenesis. The integrity of the DNA replication fork is maintained by the fork protection complex (FPC), in which TIMELESS (TIM) constitutes a key scaffold that couples the CMG helicase and replicative polymerase activities, in conjunction with its interaction with other proteins associated with the replication machinery. Loss of TIM or the FPC in general results in impaired fork progression, elevated fork stalling and breakage, and a defect in replication checkpoint activation, thus underscoring its pivotal role in protecting the integrity of both active and stalled replication forks. TIM is upregulated in multiple cancers, which may represent a replication vulnerability of cancer cells that could be exploited for new therapies. Here, we discuss recent advances on our understanding of the multifaceted roles of TIM in DNA replication and stalled fork protection, and how its complex functions are engaged in collaboration with other genome surveillance and maintenance factors.
Asunto(s)
Replicación del ADN , ADN , ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN Helicasas/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found to be associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1's sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin ß as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin ß association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1's C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (nuclear localization signal) was required for Importin ß interaction and that CT100 of Importin ß (100 amino acids of extreme C-terminus of Importin ß) was required for TopBP1 interaction. Further structure-function analysis reveals that the CTM of TopBP1 is essential for TopBP1's nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin ß-specific inhibitor importazole inhibits TopBP1's nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin ß-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1's CTM motif for ATR-Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest that the CTM motif-mediated TopBP1 shuttling into nucleus via Importin ß plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.