Structural characterization of human RPA70N association with DNA damage response proteins
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The replication protein A (RPA) plays a critical role in DNA replication, DNA repair, and recombination by interacting with various proteins. This paper describes the structure of an N-terminus OB-fold of the 70kD subunit of human replication protein A (RPA70N or DNA-binding domain-F) bound to peptides from five different proteins, HELB, ATRIP, BLM, RMI1, and WRN. This paper provides useful knowledge regarding the structural flexibility of RPA70N in the binding to the different interacting peptides. The structural and biochemical analyses of the interaction of RPA70N with the different peptides provide solid evidence for the presented conclusion. The work will be of interest to those studying DNA replication, recombination and repair.
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Abstract
The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.
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eLife assessment
The replication protein A (RPA) plays a critical role in DNA replication, DNA repair, and recombination by interacting with various proteins. This paper describes the structure of an N-terminus OB-fold of the 70kD subunit of human replication protein A (RPA70N or DNA-binding domain-F) bound to peptides from five different proteins, HELB, ATRIP, BLM, RMI1, and WRN. This paper provides useful knowledge regarding the structural flexibility of RPA70N in the binding to the different interacting peptides. The structural and biochemical analyses of the interaction of RPA70N with the different peptides provide solid evidence for the presented conclusion. The work will be of interest to those studying DNA replication, recombination and repair.
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Reviewer #1 (Public Review):
This paper describes the structure of an N-terminus OB-fold of the 70kD subunit of human replication protein A (RPA70N) bound to a peptide from five different proteins; HELB, ATRIP, BLM (two peptides), RMI1, and WRN, which are involved in various DNA transactions. This study of X-ray crystallography revealed a structural basis of RPA70N OB-fold for weak interactions with Kd of 10-18 uM. Importantly, distinct binding modes of RPA70N to different substrates indicate the flexible nature of this domain in the recognition of binding partners. In addition, the authors characterized the role of a critical hydrophobic residue in the peptides on the interactions to RPA70N by Isothermal titration calorimetry (ITC). The structural analysis of RPA70N with 6 different binding peptides is impressive and the results …
Reviewer #1 (Public Review):
This paper describes the structure of an N-terminus OB-fold of the 70kD subunit of human replication protein A (RPA70N) bound to a peptide from five different proteins; HELB, ATRIP, BLM (two peptides), RMI1, and WRN, which are involved in various DNA transactions. This study of X-ray crystallography revealed a structural basis of RPA70N OB-fold for weak interactions with Kd of 10-18 uM. Importantly, distinct binding modes of RPA70N to different substrates indicate the flexible nature of this domain in the recognition of binding partners. In addition, the authors characterized the role of a critical hydrophobic residue in the peptides on the interactions to RPA70N by Isothermal titration calorimetry (ITC). The structural analysis of RPA70N with 6 different binding peptides is impressive and the results described in the paper support the main conclusion. Understanding the structural flexibility of the RPA70N domain is important to know the molecular mechanisms of how RPA regulates distinct DNA transaction pathways. On the other hand, the authors need some in vivo functional assays to support their conclusion.
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Reviewer #2 (Public Review):
RPA is a ssDNA binding protein that functions as a hub protein to recruit more than three dozen enzyme onto DNA to coordinate almost all DNA metabolic roles. There are two specific protein interaction domain OB-F and the wh domain. NMR and crystallographic studies have solved the structure of OB-F bound to peptides from various target interactors. Nevertheless, these cognate binding sequences are not conserved. To decipher if there are unique binding modes during such interactions, Wu et. al., use a strategy to tether these target peptides to OB-F using a flexible linker and have solved the structure of complexes with peptides from HelB, ATRIP, RMI1, WRN and BLM. The high-resolution structures presented by Wu et. al. showcase key interactions between RPA70N and peptides of binding partners. These findings …
Reviewer #2 (Public Review):
RPA is a ssDNA binding protein that functions as a hub protein to recruit more than three dozen enzyme onto DNA to coordinate almost all DNA metabolic roles. There are two specific protein interaction domain OB-F and the wh domain. NMR and crystallographic studies have solved the structure of OB-F bound to peptides from various target interactors. Nevertheless, these cognate binding sequences are not conserved. To decipher if there are unique binding modes during such interactions, Wu et. al., use a strategy to tether these target peptides to OB-F using a flexible linker and have solved the structure of complexes with peptides from HelB, ATRIP, RMI1, WRN and BLM. The high-resolution structures presented by Wu et. al. showcase key interactions between RPA70N and peptides of binding partners. These findings add to similar knowledge from several other such structures that have been previously reported. The authors also suggest multivalency where multiple OB-F domains can be bound by a single peptide or a cluster of peptides. This leads to a model where such an interaction can stabilize RPA nucleoprotein filaments and better recruit interacting partners. However, such a model assumes that OB-F is floating around freely accessible to interact with other proteins. This assumption is incorrect as in most of these cases (like in Rad52-RPA interaction) there are other inter and intra protein interactions that need to be accounted for and are ignored here. Ideally, interaction studies with full length proteins provide better mechanistic understanding of interactions between proteins.
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Reviewer #3 (Public Review):
The authors have obtained beautiful structures of the OB-fold of RPA70 and peptides of interacting partners. This is accompanied by biochemical assays to show binding.
What is absent is a clear comparison of the binding sites, peptide orientations (in schematic format) and implications for regulation of ssDNA binding (by RPA70 and partner) as well as regulation of activity.
The impact of the paper in its current format is limited and can do with significant improvement.
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