Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

Abstract

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 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 β as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin β 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 β interaction and that CT100 of Importin β (100 amino acids of extreme C-terminus of Importin β) 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 β-specific inhibitor importazole inhibits TopBP1’s nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin β-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 the CTM motif-mediated TopBP1 shuttling into nucleus via Importin β plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.

The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation

“…In the previous study, the C-terminal domain of TopBP1 containing BRCT7/8 was deduced to interact with Top2␣ because Top2␣ failed to co-immunoprecipitate with a truncated TopBP1 with BRCT7/8 deleted or co-localize at UFBs with this mutant TopBP1. However, removing the C-terminal domain of TopBP1 is known to result in several cellular consequences, including mislocalization of TopBP1 and abolition of the ATR-TopBP1 interaction (29,30). One therefore cannot rule out the possibility that loss of the TopBP1-Top2␣ interaction at UFBs may be an indirect effect of interrupting a protein interaction network involving TopBP1.…”

REV1 is important for the ATR-Chk1 DNA damage response pathway in Xenopus egg extracts

“…It remains unknown, however, whether or not REV1 and ATR-Chk1 DDR pathways regulate each other and how this regulation might occur in response to ICLs in higher eukaryotes. Xenopus egg extracts has been demonstrated as an excellent cell-free model system for studies of ICL repair and DDR pathways [17,18,34]. In this communication, our compelling evidence suggests that REV1 plays a previously unidentified, but important, role in the activation of MMC-induced ATR-Chk1 DDR pathway in Xenopus egg extracts.…”

“…Caffeine, KU55933, VE-822, and NU6027 were added to egg extracts at final concentrations of 1 ng/μL, 120 μM, 10 μM, and 1 mM, respectively [10,37,38]. Aphidicolin (APH) and Mitomycin C (MMC) were added to egg extracts at final concentrations of 100 ng/μL and 0.5 mM as previously described [14,34]. Immunodepletion of REV1 was performed in a similar fashion as previously described [14].…”

Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress

“…An activated ATR kinase phosphorylates a number of downstream substrates, which are involved in nucleic acid metabolism (DNA replication, DNA repair, DNA recombination, mRNA transcription, and RNA processing), protein metabolism, and cell cycle control [83, 84]. As a critical player in DDR, Chk1 is phosphorylated at Serine 345 by ATR in response to stalled DNA replication forks and DNA damage induced by UV, IR, methyl methanesulfonate (MMS), mitomycin C (MMC), and hydrogen peroxide [85–90]. The phosphorylation of Chk1 enhances Chk1’s kinase activity, which in turn phosphorylates downstream substrates (e.g., Cdc25, BLM, and FANCD2/FANCE) and facilitates cell cycle arrest and DNA damage repair [91–93].…”