Mass spectrometric identification of lysines involved in the interaction of human replication protein a with single-stranded DNA

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.     

Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5'-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt ( approximately 19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt ( approximately 34 A). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed.     

The role for zinc in replication protein A

Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA. Modification of both the 70- and 32-kDa subunits, RPA70 and RPA32, respectively, by photoaffinity labeling indicates that RPA can bind the primer-template junction of partial duplex DNAs by interacting with the 3'-end of the primer. The identification of the protein domains modified by the photoreactive 3'-end of the primer showed that domains located in the central part of the RPA32 subunit (amino acids 39-180) and the C-terminal part of the RPA70 subunit (amino acids 432-616) are involved in these interactions.     

Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding

Although structures of single-stranded (ss)DNA-binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Angstroms structure of the ssDNA-binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules--OB-folds--which comprise the DNA-binding domain. Two OB-folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB-folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein-protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA-binding mechanism.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold

Recent years have witnessed tremendous progress in our structural and biophysical understanding of how replication protein A (RPA), a major nuclear ssDNA-binding protein (SSB), binds DNA. The four ssDNA-binding domains of RPA have the characteristic OB (oligonucleotide/oligosaccharide-binding) fold and contact DNA with specific polarity via a hierarchy-driven dynamic pathway. A growing mass of data suggest that many aspects of the ssDNA binding mechanism are conserved among SSBs of different origin. However, this conservation is not restricted to the SSB class. The concepts of ssDNA binding by the OB-fold, first derived from the RPA structure, have been successfully applied to the functional characterization of the BRCA2 (breast cancer susceptibility gene 2) protein. The BRCA2 structure, in its turn, has helped to better understand RPA function.     

The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.     

A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryarchaeal counterparts. Rather than comprising four DNA-binding domains and a zinc-finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA-binding domain in a polypeptide of just 148 amino acids with a eubacterial-like acidic C-terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein,despite possessing DNA-binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt)per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein-mediated DNA strand-exchange and rescuing the ssb-1 lethal mutation of E. coli respectively. We discuss possible evolutionary relationships amongst the various members of the SSB/RPA family.     

A new structural framework for integrating replication protein A into DNA processing machinery

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.     

Replication protein A as a major eukaryotic single-stranded DNA-binding protein and its role in DNA repair

Replication protein A (RPA) is a key regulator of eukaryotic DNA metabolism. RPA is a highly conserved heterotrimeric protein and contains multiple oligonucleotide/oligosaccharide-binding folds. The major RPA function is binding to single-stranded DNA (ssDNA) intermediates forming in DNA replication, repair, and recombination. Although binding ssDNA with high affinity, RPA can rapidly diffuse along ssDNA and destabilizes the DNA secondary structure. A highly dynamic RPA binding to ssDNA allows other proteins to access ssDNA and to displace RPA from the RPA–ssDNA complex. As has been shown recently, RPA in complex with ssDNA is posttranslationally modified in response to DNA damage. These modifications modulate the RPA interactions with its protein partners and control the DNA damage signaling pathways. The review considers up-to-date data on the RPA function as an active coordinator of ssDNA intermediate processing within DNA metabolic pathways, DNA repair in particular.     

DNA stimulates Mec1-mediated phosphorylation of replication protein A

The cellular single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) becomes phosphorylated periodically during the normal cell cycle and also in response to DNA damage. In Saccharomyces cerevisiae, RPA phosphorylation requires the checkpoint protein Mec1, a protein kinase homologous in structure and function to human ATR. We confirm here that immunocomplexes containing a tagged version of Mec1 catalyze phosphorylation of purified RPA, likely reflecting an RPA kinase activity intrinsic to Mec1. A significant stimulation of this activity is observed upon the addition of covalently closed ssDNA derived from the bacteriophage M13. This stimulation is not observed with mutant RPA deficient for DNA binding, indicating that DNA-bound RPA is a preferred substrate. Stimulation is also observed upon the addition of linear ssDNA homopolymers or hydrolyzed M13 ssDNA. In contrast to circular ssDNA, these DNA cofactors stimulate both wild type and mutant RPA phosphorylation. This finding suggests that linear ssDNA can also stimulate Mec1-mediated RPA phosphorylation by activating Mec1 or an associated protein. Although the Mec1-interacting protein Ddc2 is required for RPA phosphorylation in vivo, it is required for neither basal nor ssDNA-stimulated RPA phosphorylation in vitro. Therefore, activation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2. Our results provide insight into the mechanisms that function in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.     

Learning effective amino acid interactions through iterative stochastic techniques

The prediction of the three-dimensional structures of the native states of proteins from the sequences of their amino acids is one of the most important challenges in molecular biology. An essential task for solving this problem within coarse-grained models is the deduction of effective interaction potentials between the amino acids. Over the years, several techniques have been developed to extract potentials that are able to discriminate satisfactorily between the native and nonnative folds of a preassigned protein sequence. In general, when these potentials are used in actual dynamical folding simulations, they lead to a drift of the native structure outside the quasinative basin. In this article, we present and validate an approach to overcome this difficulty. By exploiting several numerical and analytical tools, we set up a rigorous iterative scheme to extract potentials satisfying a prerequisite of any viable potential: the stabilization of proteins within their native basin (less than 3-4 A RMSD). The scheme is flexible and is demonstrated to be applicable to a variety of parameterizations of the energy function, and it provides in each case the optimal potentials.     

Phosphorylation of replication protein A by S-phase checkpoint kinases

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3'-, but not 5'-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3'-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3'-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.     

Support Vector Machine-based classification of protein folds using the structural properties of amino acid residues and amino acid residue pairs

MOTIVATION: Fold recognition is a key step in the protein structure discovery process, especially when traditional sequence comparison methods fail to yield convincing structural homologies. Although many methods have been developed for protein fold recognition, their accuracies remain low. This can be attributed to insufficient exploitation of fold discriminatory features. RESULTS: We have developed a new method for protein fold recognition using structural information of amino acid residues and amino acid residue pairs. Since protein fold recognition can be treated as a protein fold classification problem, we have developed a Support Vector Machine (SVM) based classifier approach that uses secondary structural state and solvent accessibility state frequencies of amino acids and amino acid pairs as feature vectors. Among the individual properties examined secondary structural state frequencies of amino acids gave an overall accuracy of 65.2% for fold discrimination, which is better than the accuracy by any method reported so far in the literature. Combination of secondary structural state frequencies with solvent accessibility state frequencies of amino acids and amino acid pairs further improved the fold discrimination accuracy to more than 70%, which is approximately 8% higher than the best available method. In this study we have also tested, for the first time, an all-together multi-class method known as Crammer and Singer method for protein fold classification. Our studies reveal that the three multi-class classification methods, namely one versus all, one versus one and Crammer and Singer method, yield similar predictions. AVAILABILITY: Dataset and stand-alone program are available upon request.     

In search of the protein native state with a probabilistic sampling approach

The three-dimensional structure of a protein is a key determinant of its biological function. Given the cost and time required to acquire this structure through experimental means, computational models are necessary to complement wet-lab efforts. Many computational techniques exist for navigating the high-dimensional protein conformational search space, which is explored for low-energy conformations that comprise a protein's native states. This work proposes two strategies to enhance the sampling of conformations near the native state. An enhanced fragment library with greater structural diversity is used to expand the search space in the context of fragment-based assembly. To manage the increased complexity of the search space, only a representative subset of the sampled conformations is retained to further guide the search towards the native state. Our results make the case that these two strategies greatly enhance the sampling of the conformational space near the native state. A detailed comparative analysis shows that our approach performs as well as state-of-the-art ab initio structure prediction protocols.     

RPA and POT1

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.     

Physical interaction between replication protein A and Rad51 promotes exchange on single-stranded DNA

Replication protein A (RPA) is displaced from single-stranded DNA (ssDNA) by Rad51 during the initiation of homologous recombination. Interactions between these proteins have been reported, but the functional significance of the direct RPA-Rad51 interaction has yet to be elucidated. We have identified and characterized the interaction between DNA-binding domain A of RPA (RPA70A) and the N-terminal domain of Rad51 (Rad51N). NMR chemical shift mapping showed that Rad51N binds to the ssDNA-binding site of RPA70A, suggesting a competitive mechanism for the displacement of RPA from ssDNA by Rad51. A structure of the RPA70A-Rad51N complex was generated by experimentally guided modeling and then used to design mutations that disrupt the binding interface. Functional ATP hydrolysis assays were performed for wild-type Rad51 and a mutant defective in binding RPA. Rates of RPA displacement for the mutant were significantly below those of wild-type Rad51, suggesting that a direct RPA-Rad51 interaction is involved in displacing RPA in the initiation stage of genetic recombination.