Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

Kenta Terai,Anindya Dutta,Etsuko Shibata,Tarek Abbas

doi:10.1074/jbc.c113.505586

Abstract

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.

Highlights

The mechanism for inhibiting fork progression after DNA damage still remains
These results suggest that ubiquitination of p12 through CRL4Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression
We show that p12 has a similar PIP degron and that CRL4Cdt2 ubiquitinates and degrades p12 after UV treatment. p12 degradation is critical for inhibiting fork progression

Summary

Introduction

The mechanism for inhibiting fork progression after DNA damage still remains. Results: CRL4Cdt2 promotes the degradation of the p12. These results suggest that ubiquitination of p12 through CRL4Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression. Substrates of CRL4Cdt2, such as Cdt1, p21, and Set8, contain a threonine and a lysine in the context of the PIP box that are important for CRL4Cdt2-dependent degradation [15, 26] but not as important for PCNA binding, and the corresponding residues are present in p12 (Thr-8 and Lys-15) (Fig. 2A).

Results

Conclusion