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Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR.
Thompson MK, Ehlinger AC, Chazin WJ
(2017) Methods Enzymol 592: 49-76
MeSH Terms: DNA Primase, Humans, Multiprotein Complexes, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Domains, Replication Protein A, Scattering, Small Angle, X-Ray Diffraction
Show Abstract · Added March 24, 2018
Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional dynamics of modular multidomain proteins is a major challenge confronting structural biology today because their structure is not fixed in time. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy have proven particularly useful for the analysis of the structural dynamics of modular multidomain proteins because they provide highly complementary information for characterizing the architectural landscape accessible to these proteins. SAXS provides a global snapshot of all architectural space sampled by a molecule in solution. Furthermore, SAXS is sensitive to conformational changes, organization and oligomeric states of protein assemblies, and the existence of flexibility between globular domains in multiprotein complexes. The power of NMR to characterize dynamics provides uniquely complementary information to the global snapshot of the architectural ensemble provided by SAXS because it can directly measure domain motion. In particular, NMR parameters can be used to define the diffusion of domains within modular multidomain proteins, connecting the amplitude of interdomain motion to the architectural ensemble derived from SAXS. Our laboratory has been studying the roles of modular multidomain proteins involved in human DNA replication using SAXS and NMR. Here, we present the procedure for acquiring and analyzing SAXS and NMR data, using DNA primase and replication protein A as examples.
© 2017 Elsevier Inc. All rights reserved.
0 Communities
1 Members
0 Resources
9 MeSH Terms
Molecular basis for PrimPol recruitment to replication forks by RPA.
Guilliam TA, Brissett NC, Ehlinger A, Keen BA, Kolesar P, Taylor EM, Bailey LJ, Lindsay HD, Chazin WJ, Doherty AJ
(2017) Nat Commun 8: 15222
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Animals, Chickens, Chromatin, Crystallography, X-Ray, DNA Primase, DNA Replication, DNA-Directed DNA Polymerase, HEK293 Cells, Humans, Models, Biological, Multifunctional Enzymes, Protein Binding, Protein Domains, Replication Protein A, Xenopus
Show Abstract · Added March 24, 2018
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.
0 Communities
1 Members
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17 MeSH Terms
Identification and Optimization of Anthranilic Acid Based Inhibitors of Replication Protein A.
Patrone JD, Pelz NF, Bates BS, Souza-Fagundes EM, Vangamudi B, Camper DV, Kuznetsov AG, Browning CF, Feldkamp MD, Frank AO, Gilston BA, Olejniczak ET, Rossanese OW, Waterson AG, Chazin WJ, Fesik SW
(2016) ChemMedChem 11: 893-9
MeSH Terms: Anisotropy, Dose-Response Relationship, Drug, Fluorescence Polarization, High-Throughput Screening Assays, Models, Molecular, Molecular Structure, Replication Protein A, Structure-Activity Relationship, ortho-Aminobenzoates
Show Abstract · Added February 5, 2016
Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that initiates the DNA damage response pathway through protein-protein interactions (PPIs) mediated by its 70N domain. The identification and use of chemical probes that can specifically disrupt these interactions is important for validating RPA as a cancer target. A high-throughput screen (HTS) to identify new chemical entities was conducted, and 90 hit compounds were identified. From these initial hits, an anthranilic acid based series was optimized by using a structure-guided iterative medicinal chemistry approach to yield a cell-penetrant compound that binds to RPA70N with an affinity of 812 nm. This compound, 2-(3- (N-(3,4-dichlorophenyl)sulfamoyl)-4-methylbenzamido)benzoic acid (20 c), is capable of inhibiting PPIs mediated by this domain.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
0 Communities
1 Members
0 Resources
9 MeSH Terms
Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.
Zhao W, Vaithiyalingam S, San Filippo J, Maranon DG, Jimenez-Sainz J, Fontenay GV, Kwon Y, Leung SG, Lu L, Jensen RB, Chazin WJ, Wiese C, Sung P
(2015) Mol Cell 59: 176-87
MeSH Terms: Amino Acid Substitution, BRCA2 Protein, Breast Neoplasms, Cell Line, Female, HeLa Cells, Homologous Recombination, Humans, Models, Biological, Molecular Mimicry, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Proteasome Endopeptidase Complex, Protein Subunits, Rad51 Recombinase, Recombinant Proteins, Replication Protein A
Show Abstract · Added February 5, 2016
The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
Copyright © 2015 Elsevier Inc. All rights reserved.
1 Communities
2 Members
0 Resources
17 MeSH Terms
Functional dynamics in replication protein A DNA binding and protein recruitment domains.
Brosey CA, Soss SE, Brooks S, Yan C, Ivanov I, Dorai K, Chazin WJ
(2015) Structure 23: 1028-38
MeSH Terms: DNA, Diffusion Tensor Imaging, Gene Components, Models, Molecular, Molecular Dynamics Simulation, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Replication Protein A
Show Abstract · Added February 5, 2016
Replication Protein A (RPA) is an essential scaffold for many DNA processing machines; its function relies on its modular architecture. Here, we report (15)N-nuclear magnetic resonance heteronuclear relaxation analysis to characterize the movements of single-stranded (ss) DNA binding and protein interaction modules in the RPA70 subunit. Our results provide direct evidence for coordination of the motion of the tandem RPA70AB ssDNA binding domains. Moreover, binding of ssDNA substrate is found to cause dramatic reorientation and full coupling of inter-domain motion. In contrast, the RPA70N protein interaction domain remains structurally and dynamically independent of RPA70AB regardless of binding of ssDNA. This autonomy of motion between the 70N and 70AB modules supports a model in which the two binding functions of RPA are mediated fully independently, but remain differentially coordinated depending on the length of their flexible tethers. A critical role for linkers between the globular domains in determining the functional dynamics of RPA is proposed.
Copyright © 2015 Elsevier Ltd. All rights reserved.
0 Communities
1 Members
0 Resources
10 MeSH Terms
Simian virus Large T antigen interacts with the N-terminal domain of the 70 kD subunit of Replication Protein A in the same mode as multiple DNA damage response factors.
Ning B, Feldkamp MD, Cortez D, Chazin WJ, Friedman KL, Fanning E
(2015) PLoS One 10: e0116093
MeSH Terms: Antigens, Viral, Tumor, DNA Helicases, DNA Replication, DNA, Viral, Replication Protein A, Simian virus 40, Virus Replication
Show Abstract · Added February 4, 2016
Simian virus 40 (SV40) serves as an important model organism for studying eukaryotic DNA replication. Its helicase, Large T-antigen (Tag), is a multi-functional protein that interacts with multiple host proteins, including the ubiquitous ssDNA binding protein Replication Protein A (RPA). Tag recruits RPA, actively loads it onto the unwound DNA, and together they promote priming of the template. Although interactions of Tag with RPA have been mapped, no interaction between Tag and the N-terminal protein interaction domain of the RPA 70kDa subunit (RPA70N) has been reported. Here we provide evidence of direct physical interaction of Tag with RPA70N and map the binding sites using a series of pull-down and mutational experiments. In addition, a monoclonal anti-Tag antibody, the epitope of which overlaps with the binding site, blocks the binding of Tag to RPA70N. We use NMR chemical shift perturbation analysis to show that Tag uses the same basic cleft in RPA70N as multiple of DNA damage response proteins. Mutations in the binding sites of both RPA70N and Tag demonstrate that specific charge reversal substitutions in either binding partner strongly diminish the interaction. These results expand the known repertoire of contacts between Tag and RPA, which mediate the many critical roles of Tag in viral replication.
1 Communities
4 Members
0 Resources
7 MeSH Terms
High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.
Bhat KP, Bétous R, Cortez D
(2015) J Biol Chem 290: 4110-7
MeSH Terms: Amino Acid Motifs, Bone Neoplasms, DNA Damage, DNA Helicases, DNA Replication, DNA, Single-Stranded, Electrophoretic Mobility Shift Assay, Humans, Osteosarcoma, Protein Binding, Replication Protein A, Substrate Specificity, Tumor Cells, Cultured
Show Abstract · Added January 20, 2015
SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
0 Communities
1 Members
0 Resources
13 MeSH Terms
Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.
Guilliam TA, Jozwiakowski SK, Ehlinger A, Barnes RP, Rudd SG, Bailey LJ, Skehel JM, Eckert KA, Chazin WJ, Doherty AJ
(2015) Nucleic Acids Res 43: 1056-68
MeSH Terms: DNA Primase, DNA Primers, DNA Replication, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Humans, Mitochondrial Proteins, Multifunctional Enzymes, Mutagenesis, Proliferating Cell Nuclear Antigen, Protein Interaction Domains and Motifs, Replication Protein A
Show Abstract · Added January 20, 2015
PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.
© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
1 Communities
3 Members
0 Resources
12 MeSH Terms
Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1.
Feldkamp MD, Mason AC, Eichman BF, Chazin WJ
(2014) Biochemistry 53: 3052-61
MeSH Terms: Amino Acid Sequence, Crystallography, X-Ray, DNA Helicases, Models, Molecular, Replication Protein A, Sequence Alignment
Show Abstract · Added May 19, 2014
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.
1 Communities
2 Members
0 Resources
6 MeSH Terms
Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A.
Frank AO, Vangamudi B, Feldkamp MD, Souza-Fagundes EM, Luzwick JW, Cortez D, Olejniczak ET, Waterson AG, Rossanese OW, Chazin WJ, Fesik SW
(2014) J Med Chem 57: 2455-61
MeSH Terms: Alanine, Amino Acid Sequence, Cell Line, Crystallization, Crystallography, X-Ray, DNA, Single-Stranded, Drug Discovery, Electrophoretic Mobility Shift Assay, Fluorescence Polarization, Magnetic Resonance Spectroscopy, Microscopy, Fluorescence, Models, Molecular, Molecular Sequence Data, Penetrance, Peptides, Protein Conformation, Replication Protein A, Structure-Activity Relationship, Tumor Suppressor Protein p53
Show Abstract · Added March 11, 2014
Stapled helix peptides can serve as useful tools for inhibiting protein-protein interactions but can be difficult to optimize for affinity. Here we describe the discovery and optimization of a stapled helix peptide that binds to the N-terminal domain of the 70 kDa subunit of replication protein A (RPA70N). In addition to applying traditional optimization strategies, we employed a novel approach for efficiently designing peptides containing unnatural amino acids. We discovered hot spots in the target protein using a fragment-based screen, identified the amino acid that binds to the hot spot, and selected an unnatural amino acid to incorporate, based on the structure-activity relationships of small molecules that bind to this site. The resulting stapled helix peptide potently and selectively binds to RPA70N, does not disrupt ssDNA binding, and penetrates cells. This peptide may serve as a probe to explore the therapeutic potential of RPA70N inhibition in cancer.
0 Communities
4 Members
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19 MeSH Terms