最先端研究セミナー

 

Abstract

During a cell division cycle, both genetic and epigenetic informations must be correctly transmitted into daughter cells. Control of DNA replication-licensing ensures that chromosomal DNA is replicated once-and-only once in a cell cycle. By the early G1 phase, Cdc6 and Cdt1 load DNA helicase, MCM2-7 on replication origins for licensing. We reported that high expression of Cdt1 and/or Cdc6 caused over-replication of DNA, demonstrating that control of licensing factors is essential for only once replication. When cells started S phase, PCNA is loaded on chromatin and connects Cdt1 to ubiquitin ligase CRL4(Cul4-DDB1)-Cdt2 for degradation, thus preventing the re-replication. Cdt1 associates through its PIP-box (PCNA interacting protein box) to PCNA and is recognized by Cdt2. When cells are exposed to DNA damaging agents including UV-irradiation, the same PCNA and CRL4-Cdt2 mediated pathway is activated in G1 phase to degrade Cdt1, which appears to be important for repair of DNA damages. In addition to Cdt1, several targets of CRL4-Cdt2 have been reported, including H4K20 monomethylase Set8, CDK inhibitor p21, and thymine DNA glycosylase (TDG); all these substrate proteins are directly or indirectly involved in replication licensing or genome integrity. Recently, we found that human Cdt2 has a PIP-box at its C-terminal end. This motif is important to recruiting CRL4-Cdt2 to PCNA sites for efficient Cdt1 degradation. We propose that direct binding of Cdt2 to PCNA is important for targeting the CRL4^Cdt2 E3 ligase activity to substrates to maintain genome integrity.

 

References

1. Nukina K, et. al. Mutations at multiple CDK-phosphorylation consensus sites on Cdt2 increase the affinity of CRL4^Cdt2 for PCNA and its ubiquitination activity in S phase. Genes to Cells (in press)
2. Tanaka M, et. al. Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase. Cell Cycle 16(7):673-684 (2017)
3. Shiomi Y and Nishitani H. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication. Genes (Basel). 8(2). pii: E52 (2017)
4. Morino M, et. al. Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response. PLoS One. 10(3):e0120553 (2015)
5. Hayashi A, et. al. PCNA-dependent ubiquitination of Cdt1 and p21 in mammalian cells. Methods Mol Biol. 1170:367-82 (2014)
6. Shiomi Y, et. al. Two different RFC proteins, Ctf18 and RFC1 separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV-irradiation. Mol Cell Biol. 32(12):2279-88 (2012).
7. Nishitani H, et. al. Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. EMBO J. 25(5):1126-36 (2006)
8. Nishitani H, et. al. The cdt1 protein is required to license DNA for replication in fission yeast. Nature. 404(6778):625-628 (2000)

 

担当分野：　損傷修復　立石（内線：6605）

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