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

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, phosphoNbs1 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 singlestranded 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 singlestranded 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.Both replicative stress and DNA damage initiate cellular processes collectively termed the DNA damage response. These processes include activation of appropriate DNA repair mechanisms, delay of the cell cycle in order to allow sufficient time for repair, and in some cases apoptosis (1, 2). One hallmark of the DNA damage response is the aggregation of multiprotein complexes into foci or repair centers. The composition of the foci depends upon the nature of the DNA lesion and is temporally dynamic, changing as the damage is first recognized, processed, and then repaired (3, 4). The assembly of foci appears to be largely governed by a network of protein-protein interactions rather than DNA-protein interactions (3, 4), providing an explanation for the dynamic composition of the foci. Although different types of DNA damage activate specific repair pathways and therefore specific proteins, there are some protein factors that respond to multiple types of lesions (3-6). These global response proteins are organized in a distinct spatiotemporal fashion depending upon the nature of the DNA lesion (3, 4). Two protein complexes that are intimately involved in the DNA damage response to multiple types of lesions are the Mre11/Rad50/Nbs1 (MRN) 1 complex and replication protein A (RPA).The MRN complex is best known for its role in DNA doublestrand break (DSB) repair (7), but more recent evidence has expanded this role to include participation in DNA replication, cell cycle checkpoints, telomere maintenance, signa...

Cell Cycle- and Proteasome-Dependent Formation of Etoposide-Induced Replication Protein A (RPA) or Mre11/Rad50/Nbs1 (MRN) Complex Repair Foci

“…65 We previously reported that RPA and MRN complex co-localization is dependent on the type of damage present. 21 We observed that treatment with ETOP led to cells containing either RPA or phospho-Nbs1 foci, but not both. In the companion paper, we demonstrate that RPA is however required for the formation of the MRN foci.…”

“…4,[10][11][12][13][14][15][16][17][18][19][20][21] RPA is also hyperphosphorylated (particularly the p34 subunit) following DNA damage and replicative stress. [20][21][22][23] which is hypothesized to function as a "switch" to direct RPA activity from DNA replication to DNA repair. 9,24,25 The MRN complex is composed of Mre11, Rad50 and Nbs1.…”

Nucleoside analogs: molecular mechanisms signaling cell death

“…A similar phenomenon is evident in response to stalled replication forks. Nuclear co-localization of Mre11, Rad50 and Nbs1 with other DNA damage response molecules, phosphorylated ATM and H2AX, increases in response to gemcitabine, ara-C, troxacitabine and hydroxyurea (Wang et al, 2000;Mirzoeva and Petrini, 2003;Robison et al, 2005;Ewald et al, 2008).…”

Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins

“…What is the biological significance underlying the specific binding of ScMre11 to intermediates of recombination and repair? A large body of evidence supports the view that the Mre11 complex plays a key role in the process of DNA replication (65)(66)(67)(68)(69)(70); however, the mechanism is less well characterized. The observation that Mre11 exhibits striking selectivity toward replication fork and flayed duplex is consistent with its demonstrated role during "normal" replication or under conditions of replicative stress (70).…”

“…Moreover, recent studies have shown that Mre11 complex, specifically its nuclease activity, plays an essential role in processing of the stalled replication forks, thereby helping to maintain genome integrity during replication stress (65)(66)(67)(68)(69)(70). To understand the mechanism of processing of stalled replication forks and other intermediates by MRX complex better, we considered whether ScMre11 could unwind replication and recombination intermediates, such as the Holliday junction and flayed duplex substrates.…”