Regulation of PCNA polyubiquitination in human cells

Jan Brun,Douglas A Gray,Bradly G Wouters,Roland K Chiu

doi:10.1186/1756-0500-3-85

Abstract

BackgroundThe ubiquitin-based molecular switch dictating error free versus error prone repair has been conserved throughout eukaryotic evolution. A central component of this switch is the homotrimeric clamp PCNA, which is ubiquitinated in response to genotoxic stress allowing recovery of replication forks blocked at sites of DNA damage. The particulars of PCNA ubiquitination have been elucidated in yeast and to a further extent recently in human cells. However, gaps in the detailed mechanism and regulation of PCNA polyubiquitination still persist in human cells.FindingsWe expand upon several studies and show that PCNA is polyubiquitnated in normal skin fibroblasts, and that this ubiquitination is dependant on RAD18. Furthermore we define the types of DNA damage that induce ubiquitination on PCNA. Cisplatin, methylmethane sulphonate and benzo(a)pyrene-diol-epoxide induce the polyubiquitination of PCNA to the same extent as UV while polyubiquitination is not detected after X-ray treatment. Moreover, we show that ubiquitination of PCNA is not regulated by cell cycle checkpoint kinases ATM-Chk2 or ATR-Chk1. Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.ConclusionsOur results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination.

Highlights

In recent studies proliferating cell nuclear antigen (PCNA) ubiquitination has been identified as an important modification in human cells [1,2,3,4]
Our results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination
In a previous study we were the first to demonstrate that K63linked polyubiquitination is important in human fibroblasts and that PCNA is both mono and polyubiquitinated in cancer cell lines by Rad18 and Ubc13 [1]

Summary

Introduction

In recent studies PCNA ubiquitination has been identified as an important modification in human cells [1,2,3,4]. The use of cancer cells may result in a distortion of the physiological damage response It remains to be determined whether PCNA polyubiquitination is important in. Another central question is how human cells regulate mono and polyubiquitination of PCNA and how they induce or limit the deployment of DNA repair machinery in the presence or absence of damage. One possibility is that PCNA ubiquitination is regulated by cell cycle checkpoint kinases ATR-Chk or ATM-Chk given their central role in damage surveillance [8,9,10,11]. Since cell cycle checkpoint kinases are predominantly activated after DNA damage we sought to determine whether PCNA ubiquitination is regulated by global sensors such as ATR or ATM. We confirm that the candidate DUB for negatively regulating PCNA polyubiquitination is USP1

Experimental Procedures

Results

Discussion