Takuya Abe, Ryotaro Kawasumi, Michele Giannattasio, Sabrina Dusi, Yui Yoshimoto, Keiji Miyata, Koyuki Umemura, Kouji Hirota & Dana Branzei
Nature Communications volume 9, Article number: 3091 (2018)
Abstract
AND-1/Ctf4 bridges the CMG helicase and DNA polymerase alpha, facilitating replication. Using an inducible degron system in avian cells, we find that AND-1 depletion is incompatible with proliferation, owing to cells accumulating in G2 with activated DNA damage checkpoint. Replication without AND-1 causes fork speed slow-down and accumulation of long single-stranded DNA (ssDNA) gaps at the replication fork junction, with these regions being converted to DNA double strand breaks (DSBs) in G2. Strikingly, resected forks and DNA damage accumulation in G2, but not fork slow-down, are reverted by treatment with mirin, an MRE11 nuclease inhibitor. Domain analysis of AND-1 further revealed that the HMG box is important for fast replication but not for proliferation, whereas conversely, the WD40 domain prevents fork resection and subsequent DSB-associated lethality. Thus, our findings uncover a fork protection function of AND-1/Ctf4 manifested via the WD40 domain that is essential for proliferation and averts genome instability.
Author information
Author notes
These authors contributed equally: Takuya Abe, Ryotaro Kawasumi.
Nature Communications volume 9, Article number: 3091 (2018)
AND-1/Ctf4 bridges the CMG helicase and DNA polymerase alpha, facilitating replication. Using an inducible degron system in avian cells, we find that AND-1 depletion is incompatible with proliferation, owing to cells accumulating in G2 with activated DNA damage checkpoint. Replication without AND-1 causes fork speed slow-down and accumulation of long single-stranded DNA (ssDNA) gaps at the replication fork junction, with these regions being converted to DNA double strand breaks (DSBs) in G2. Strikingly, resected forks and DNA damage accumulation in G2, but not fork slow-down, are reverted by treatment with mirin, an MRE11 nuclease inhibitor. Domain analysis of AND-1 further revealed that the HMG box is important for fast replication but not for proliferation, whereas conversely, the WD40 domain prevents fork resection and subsequent DSB-associated lethality. Thus, our findings uncover a fork protection function of AND-1/Ctf4 manifested via the WD40 domain that is essential for proliferation and averts genome instability.
Author information
Author notes
These authors contributed equally: Takuya Abe, Ryotaro Kawasumi.
Affiliations
IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139, Milan, Italy
Takuya Abe, Ryotaro Kawasumi, Michele Giannattasio, Sabrina Dusi & Dana Branzei
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan
Takuya Abe, Yui Yoshimoto, Keiji Miyata, Koyuki Umemura & Kouji Hirota
Dipartimento di Oncologia ed Emato-Oncologia, Università degli Studi di Milano, Milan, 20122, Italy
Michele Giannattasio
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Via Abbiategrasso 207, 27100, Pavia, Italy
Dana Branzei
Contributions
T.A., R.K., and D.B. designed the research; T.A. and R.K. performed the experiments; M.G. and S.D. performed TEM sample acquisition and analysis; Y.Y., K.M., K.U., and K.H. contributed to experiments on molecular combing and pulse field gel electrophoresis; T.A., R.K., and D.B analyzed the data; T.A. and R.K. made the figures; D.B. wrote the paper and all authors contributed suggestions.
Competing interests
The authors declare no competing interests.
Corresponding author
Correspondence to Dana Branzei.
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