RBX1 (RING Box Protein 1) E3 Ubiquitin Ligase Is Required for Genomic Integrity by Modulating DNA Replication Licensing Proteins

Lijun Jia,Jeremy S Bickel,Jiaxue Wu,Meredith A Morgan,Hua Li,Jie Yang,Xiaochun Yu,Raymond C Chan,Yi Sun

doi:10.1074/jbc.m110.188425

Abstract

RBX1 (RING box protein 1), also known as ROC1 (Regulator of Cullin 1), is an essential component of SCF (Skp1/Cullins/F-box) E3 ubiquitin ligases, which target diverse proteins for proteasome-mediated degradation. Our recent study showed that RBX1 silencing triggered a DNA damage response (DDR) leading to G(2)-M arrest, senescence, and apoptosis, with the mechanism remaining elusive. Here, we show that, in human cancer cells, RBX1 silencing causes the accumulation of DNA replication licensing proteins CDT1 and ORC1, leading to DNA double-strand breaks, DDR, G(2) arrest, and, eventually, aneuploidy. Whereas CHK1 activation by RBX1 silencing is responsible for the G(2) arrest, enhanced DNA damage renders cancer cells more sensitive to radiation. In Caenorhabditis elegans, RBX-1 silencing causes CDT-1 accumulation, triggering DDR in intestinal cells, which is largely abrogated by simultaneous CDT-1 silencing. RBX-1 silencing also induces lethality during development of embryos and in adulthood. Thus, RBX1 E3 ligase is essential for the maintenance of mammalian genome integrity and the proper development and viability in C. elegans.

Highlights

We demonstrate that RBX1 silencing causes DNA double-strand breaks (DSB), leading to chromosome aneuploidy, 4 The abbreviations used are: DDR, DNA damage response(s); DSB, doublestrand break(s); siRBX1, RBX1 siRNA; siCONT, control siRNA
RBX1 Silencing Induces DDR as a Result of DNA DSB—We recently reported that RBX1 silencing induces growth arrest at the G2-M phase and cell death via senescence and apoptosis, which are coupled with the phosphorylation of H2AX, an indicator of DDR [19]
To further characterize the DDR upon RBX1 silencing, we first determined the levels of CHK1 and CHK2 phosphorylation, two classical DDR markers, and found that both were significantly induced upon RBX1 silencing in H1299 and U87 cancer cell lines (Fig. 1, A and B)

Summary

Introduction

We performed a neutral comet assay to determine whether RBX1 silencing causes DNA DSB that may trigger the observed DDR and found that comet tail moment (a measurement of DSB) was significantly greater in RBX1 siRNA-transfected cells than in control siRNAtransfected cells (Fig. 1D). RBX1 Silencing Induces the Accumulation of CDT1 and ORC1, Two DNA Replication Licensing Proteins—Previous studies have shown that DNA replication aberrance, followed by activation of the DDR, could be triggered by the activation of oncogenes or the dysregulation of DNA replication licensing proteins (29 –31). RBX1 silencing abrogates SCF E3 ligase activity and causes CDT1/ORC1 accumulation, which likely triggers DSB and DDR as demonstrated by a recent report that

Results

Conclusion