DNA Lesion-specific Co-localization of the Mre11/Rad50/Nbs1 (MRN) Complex and Replication Protein A (RPA) to Repair Foci

Jacob G Robison,Lu Lu,Kathleen Dixon,John J Bissler

doi:10.1074/jbc.m414391200

Abstract

The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci were detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions.

Highlights

Both replicative stress and DNA damage initiate cellular processes collectively termed the DNA damage response
We have previously shown that the MRN complex and replication protein A (RPA) co-localize to stalled replication forks where they interact following replicative stress induced by hydroxyurea (HU) or UV [15]
The loss of co-localization in the prolonged HU-treated cells together with DNA doublestrand break (DSB) induced by ETOP and mitomycin C (MMC), leading to a very small percentage of focipositive cells expressing both phospho-Nbs1 and RPA foci, suggests that the MRN complex and RPA respond to DNA DSBs independently of one another

Summary

Introduction

Both replicative stress and DNA damage initiate cellular processes collectively termed the DNA damage response. The MRN complex forms foci at sites of DNA replication, DNA damage, and DNA repair [3, 15,16,17,18,19,20] Both Mre and Nbs become phosphorylated following treatment of cells with genotoxic agents [21, 22], and Mre is phosphorylated in a DNA replication-dependent manner [10]. We wanted to determine whether the interaction of the MRN complex with RPA was specific to replicative stress responses or whether other types of DNA damage stimulate the interaction. To achieve this goal, we used topoisomerase inhibitors and cross-linking agents.

Methods

Results

Conclusion