Replication Protein A and the Mre11·Rad50·Nbs1 Complex Co-localize and Interact at Sites of Stalled Replication Forks

Abstract

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or -phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU-or UVinduced replication stress and suggest that protein phosphorylation may play a role in this interaction.Stalled replication forks threaten DNA replication fidelity and genomic integrity. Stalled forks occur after deficiencies in replication substrates, inhibition of replication machinery, or blocking of the replication machinery by DNA damage or DNAprotein complexes (1, 2). Replication fork stalling with subsequent replication stress is also thought to occur during normal DNA replication, particularly at DNA sequences that are prone to form secondary structures (e.g. tRNA genes) or in regions where transcription complexes may collide (2). Failure to resolve this replication stress may result in the collapse of stalled forks and genomic instability (3). To prevent such instability, replication stress triggers the activation of a DNA damage response. This response involves the recruitment and activation of proteins involved in DNA repair and cell cycle regulation. This DNA damage response uses proteins to detect damage, signal the site of damage, and transduce and amplify the signal to activate needed effector proteins. Many proteins involved in these three aspects accumulate and form large nuclear foci after DNA damage. Examples of such proteins include ␥H2AX, 53BP1, ATM, ATR, BRCA1, Werner's protein, the MRN 1 complex, and RPA1 (4, 5). The functions of these foci are not fully understood, but they may represent site...

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

“…71 A phospho-specific antibody for Nbs1 is known to be damage-dependent, and we previously showed that the phospho-RPA antibody is specific for the damage-induced hyperphosphorylated form of RPA-p34. 20 ETOP treatment led to the increase in all three phospho-proteins investigated as expected regardless of cell cycle phase (Fig. 4).…”

“…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

“…Dysfunction of ATM or the MRN complex subunits results in embryonic lethality in eukaryotes (Xiao and Weaver, 1997;Luo et al, 1999;Zhu et al, 2001a) and hypomorphic mutations are associated with a variety of human disorders, including ataxia-telangiectasia (AT), ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS, Carney et al, 1998;Matsuura et al, 1998;Varon et al, 1998;Stewart et al, 1999;Shiloh, 2006), suggesting their involvement during normal DNA replication. At the molecular level, the MRN complex associates with chromatin in an S phasespecific manner (Mirzoeva and Petrini, 2001) and binds with RPA (Robison et al, 2004;Olson et al, 2007). A similar phenomenon is evident in response to stalled replication forks.…”

Nibrin functions in Ig class-switch recombination

“…The nibrin͞Mre11͞Rad50 complex could be targeted to sites of action of AID by means of replication protein A, which can bind to Mre11 (29), and to AID (30). So far, the exact function of nibrin in class-switch recombination is not clear.…”