Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1.
Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.
Oakley
Greg G
GG
College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska 68583, USA.
Tillison
Kristin
K
Opiyo
Stephen A
SA
Glanzer
Jason G
JG
Horn
Jeffrey M
JM
Patrick
Steve M
SM
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D000076228:MRE11 Homologue Protein; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000378:metabolism*; D051716:Replication Protein A / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; C497167:RPA1 protein, human; D051716:Replication Protein A; C516794:RPA2 protein, human; D000076228:MRE11 Homologue Protein