An Evolutionarily Conserved Function of Proliferating Cell Nuclear Antigen for Cdt1 Degradation by the Cul4-Ddb1 Ubiquitin Ligase in Response to DNA Damage

Jian Hu,Yue Xiong

doi:10.1074/jbc.c500464200

Abstract

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF(Skp2), evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage.

Highlights

Cdt1, first identified in the fission yeast Schizosaccharomyces pombe as a G1 START component Cdc10-dependent transcript whose loss-of-function prevents DNA replication [1], binds to the origin recognition complex with Cdc6 at the origin of replication and together with Cdc6 and origin recognition complex recruits the minichromosome maintenance 2–7 (MCM2–7) to assemble the prereplication complexes during G1, thereby controlling the initiation of DNA replication [2]
The major difference between these two CDK inhibitors lies in their C-terminal regions; while p21 contains a proliferating cell nuclear antigen (PCNA) binding and inhibitory domain in its C-terminal region, the C-terminal sequence of p27 plays roles in regulating the phosphorylation of p27 and stability but has no PCNA binding activity
This prompted us to determine which region in p21, the N-terminal CDKcyclin binding or C-terminal PCNA binding sequence, is required for inhibiting Cdt1 degradation following UV irradiation

Summary

Introduction

Cdt1, first identified in the fission yeast Schizosaccharomyces pombe as a G1 START component Cdc10-dependent transcript whose loss-of-function prevents DNA replication [1], binds to the origin recognition complex with Cdc6 at the origin of replication and together with Cdc6 and origin recognition complex recruits the minichromosome maintenance 2–7 (MCM2–7) to assemble the prereplication complexes during G1, thereby controlling the initiation of DNA replication [2]. To determine how CDK activity may affect Cdt1 degradation in response to DNA damage, we overexpressed the CDK inhibitors p21 and p27 in HEK 293T cells and determined the steady-state levels of Cdt1 protein following UV irradiation.

Results

Conclusion