Proliferating Cell Nuclear Antigen Is Protected from Degradation by Forming a Complex with MutT Homolog2

Yu Yu,Jian-Ping Cai,Bo Tu,Lipeng Wu,Ying Zhao,Xiangyu Liu,Lian Li,Michael A Mcnutt,Jingnan Feng,Qihua He,Yang Yang,Haiying Wang,Mutsuo Sekiguchi,Wei-Guo Zhu

doi:10.1074/jbc.m109.015289

Abstract

Proliferating cell nuclear antigen (PCNA) has been demonstrated to interact with multiple proteins involved in several metabolic pathways such as DNA replication and repair. However, there have been fewer reports about whether these PCNA-binding proteins influence stability of PCNA. Here, we observed a physical interaction between PCNA and MutT homolog2 (MTH2), a new member of the MutT-related proteins that hydrolyzes 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP). In several unstressed human cancer cell lines and in normal human fibroblast cells, PCNA and MTH2 formed a complex and their mutual binding fragments were confirmed. It was intriguing that PCNA and MTH2 were dissociated dependent on acetylation of PCNA, which in turn induced degradation of PCNA in response to UV irradiation, but not in response to other forms of DNA-damaging stress. To further explore the link between dissociation of PCNA-MTH2 and degradation of PCNA, RNAi against MTH2 was performed to mimic the dissociated status of PCNA to evaluate changes in the half-life of PCNA. Knockdown of MTH2 significantly promoted degradation of PCNA, suggesting that the physiological interaction of PCNA-MTH2 may confer protection from degradation for PCNA, whereas UV irradiation accelerates PCNA degradation by inducing dissociation of PCNA-MTH2. Moreover, secondary to degradation of PCNA, UV-induced inhibition of DNA synthesis or cell cycle progression was enhanced. Collectively, our data demonstrate for the first time that PCNA is protected by this newly identified partner molecule MTH2, which is related to DNA synthesis and cell cycle progression.

Highlights

Because Proliferating cell nuclear antigen (PCNA) is essential for DNA synthesis both in DNA replication and repair, a dynamic balance between PCNA synthesis and degradation is critical for maintaining normal DNA synthesis
RibA is a backup enzyme for MutT in E. coli and plays a role in maintaining high fidelity of phate; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; TSA, trichostatin A; ROI, region of interest; Fluorescence Resonance Energy Transfer (FRET), fluorescence resonance energy transfer; PIPES, 1,4-piperazinediethanesulfonic acid; RNA Interference (RNAi), RNA interference; CHX, cycloheximide; TLS, translesion synthesis; RT, reverse transcription
It was of interest to observe that PCNA and MutT homolog2 (MTH2) formed a complex in vivo (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Streptomycin in a 37 °C, 5% CO2, humidified incubator. Human lung diploid fibroblast cells (2BS) or human embryonic kidney 293T cells (HEK293T) were grown in Dulbecco’s modified Eagle’s medium (10% fetal bovine serum). F and G, both PCNA and MTH2 were detected by Western blotting (F) or RT-PCR (G) in A549 cells irradiated with UV at different doses or for different incubation times after irradiation. Equal amounts of GST or GST fusion proteins were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) by rocking at 4 °C for 1 h, and the beads were washed three times with TEN buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, and 100 mM NaCl). The beads were washed three times with TENT buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, and 300 mM NaCl), and dissolved into 2ϫ SDS loading buffer after centrifugation and boiled 5 min at 100 °C. The supernatant was extracted and analyzed by Western blotting with antibodies to PCNA and MTH2. To examine the efficiency of siRNA, total protein was extracted for Western blotting using anti-MTH2 or anti-PCNA. The fluorescence value was quantified with TCS SP2 confocal software

RESULTS

To investigate whether the decrease in interaction between

DISCUSSION