Replication Protein A Protein Research Articles

Lnk/Sh2b3 Deficiency Increases DNA Damage Tolerance to Promote Hematopoietic Stem Cell Fitness

DNA repair deficiency depicted by the syndrome Fanconi Anemia (FA), results in progressive bone marrow failure (BMF), increased leukemia incidence and cancer susceptibility. Out of the 23 causative genes for FA, FANCD2 is crucial for its central role in the regulation of DNA repair and replication. The FA pathway plays an important role in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis and genome integrity. Our previous studies revealed that loss of the adaptor protein LNK (SH2B3), a negative regulator of cytokine signaling, restores hematopoietic stem cell (HSC) function and genome integrity in Fancd2 deficient FA mouse models and primary human FA progenitors. Notably, mice deficient in Lnk harbor a remarkable 14-fold expansion of HSCs with superior self-renewal capability. However, the protective mechanisms that LNK controls to prevent stem cell attrition and increase stem cell fitness are not established. Here, we show that Lnk deficiency reduced DNA damage in Fancd2-/- HSCs at the steady-state in vivo, as well as in forced proliferation upon transplantation. Lnk deficiency reduced HSPC DNA damage in replication stress upon cytokine-induced proliferation in ex vivo culture, accompanied by reduced p53 activation. One injection of pIpC induces HSPC into the cell cycle within 24hr, thus we used this as a model to study replication stress in vivo. We found that Lnk deficiency reduced ATR but not ATM pathway activation in Fancd2-/- HSCs in pIpC-induced proliferation in vivo. ATR is activated by single-strand DNA breaks (SSBs) during replication stress; thus we directly examined the amount of single-strand DNA (ssDNA) and the level of ssDNA binding protein RPA (Replication Protein A) in HSCs during the cell cycle. Of note, Lnk deficiency reduced ssDNA as well as chromatin-bound RPA2 in Fancd2-/- HSCs in replication in vivo. Lnk deficiency also reduced RPA2 phosphorylation in HSPCs under Camptothecin (CPT) and hydroxyurea (HU) induced replication stress conditions. Mechanistically, loss of Lnk protected stalled replication fork from degradation, suppressed ssDNA gaps, and promoted replication fork restart in Fancd2-/- HSPCs upon HU-induced replication stress. In light of our data suggesting a new role for LNK in replication stress-induced DNA damage, we examined the DNA damage tolerance (DDT) mechanism that enables DNA replication to circumvent the lesions, thereby allowing the completion of DNA replication, increasing the survival of HSPCs and preventing genome instability. One of the major DDT pathways is translesion synthesis (TLS) in which the replicative DNA polymerase is temporarily replaced by special TLS polymerases pol ζ or η that can replicate across DNA lesions. Indeed, we found that Lnk deficient HSCs have increased chromatin-bound TLS polymerase eta (Pol ƞ) and were resistant to shRNA-mediated knockdown of PolH (the gene encodes polymerase eta) compared to wildtype HSCs in transplant assays. Strikingly, Lnk deficient HSCs were more resistant to the inhibitor of Rev1 that disrupts Rev1/ pol ζ interaction than wildtype HSCs in transplanting assays. These findings highlight a previously-unappreciated role of LNK in limiting DNA damage tolerance mechanisms in both normal and diseased replicative states. The novel link between LNK-controlled cytokine signaling and TLS sheds light on our understanding of replication stress mitigation to promote HSC fitness that can be explored for future regeneration medicine.

Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding.

Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level. In the past three decades, there has been a large increase in the application of single-molecule approaches to investigate helicases. These techniques, such as optical tweezers and single-molecule fluorescence, offer capabilities to monitor helicase motions with unprecedented spatiotemporal resolution, to apply quantitative forces to probe the chemo-mechanical activities of these motors and to resolve helicase heterogeneity at the single-molecule level. In this chapter, we describe a single-molecule method that combines optical tweezers with confocal fluorescence microscopy to study helicase-catalyzed DNA unwinding. Using Bloom syndrome protein (BLM), a multifunctional helicase that maintains genome stability, as an example, we show that this method allows for the simultaneous detection of displacement, force and fluorescence signals of a single DNA molecule during unwinding in real time, leading to the discovery of a distinct bidirectional unwinding mode of BLM that is activated by a single-stranded DNA binding protein called replication protein A (RPA). We provide detailed instructions on how to prepare two DNA templates to be used in the assays, purify the BLM and RPA proteins, perform single-molecule experiments, and acquire and analyse the data.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.

Repair of DNA double‐stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post‐translational modification of repair factors. In budding yeast, single‐stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA–RPA complexes are then SUMO‐modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single‐stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO‐RPA signal is amplified by successive rounds of Siz2‐dependent SUMO modification of RPA and dissociation of SUMO‐RPA at the junction between single‐ and double‐stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.

Remote Homology Detection Identifies a Eukaryotic RPA DBD-C-like DNA Binding Domain as a Conserved Feature of Archaeal Rpa1-Like Proteins.

The eukaryotic single-stranded DNA binding factor replication protein A (RPA) is essential for DNA replication, repair and recombination. RPA is a heterotrimer containing six related OB folds and a winged helix-turn-helix (wH) domain. The OB folds are designated DBD-A through DBD-F, with DBD-A through DBD-D being directly involved in ssDNA binding. DBD-C is located at the C-terminus of the RPA1 protein and has a distinctive structure that includes an integral C4 zinc finger, while the wH domain is found at the C-terminus of the RPA2 protein. Previously characterised archaeal RPA proteins fall into a number of classes with varying numbers of OB folds, but one widespread class includes proteins that contain a C4 or C3H zinc finger followed by a 100–120 amino acid C-terminal region reported to lack detectable sequence or structural similarity. Here, the sequences spanning this zinc finger and including the C-terminal region are shown to comprise a previously unrecognised DBD-C-like OB fold, confirming the evolutionary relatedness of this group of archaeal RPA proteins to eukaryotic RPA1. The evolutionary relationship between eukaryotic and archaeal RPA is further underscored by the presence of RPA2-like proteins comprising an OB fold and C-terminal winged helix (wH) domain in multiple species and crucially, suggests that several biochemically characterised archaeal RPA proteins previously thought to exist as monomers are likely to be RPA1-RPA2 heterodimers.

Revisiting regulatory roles of replication protein A in plant DNA metabolism.

This review provides insight into the roles of heterotrimeric RPA protein complexes encompassing all aspects of DNA metabolism in plants along with specific function attributed by individual subunits. It highlights research gaps that need further attention. Replication protein A (RPA), a heterotrimeric protein complex partakes in almost every aspect of DNA metabolism in eukaryotes with its principle role being a single-stranded DNA-binding protein, thereby providing stability to single-stranded (ss) DNA. Although most of our knowledge of RPA structure and its role in DNA metabolism is based on studies in yeast and animal system, in recent years, plants have also been reported to have diverse repertoire of RPA complexes (formed by combination of different RPA subunit homologs arose during course of evolution), expected to be involved in plethora of DNA metabolic activities. Here, we have reviewed all studies regarding role of RPA in DNA metabolism in plants. As combination of plant RPA complexes may vary largely depending on number of homologs of each subunit, next step for plant biologists is to develop specific functional methods for detailed analysis of biological roles of these complexes, which we have tried to formulate in our review. Besides, complete absence of any study regarding regulatory role of posttranslational modification of RPA complexes in DNA metabolism in plants, prompts us to postulate a hypothetical model of same in light of information from animal system. With our review, we envisage to stimulate the RPA research in plants to shift its course from descriptive to functional studies, thereby bringing a new angle of studying dynamic DNA metabolism in plants.

Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1.

Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.

Distinct expression and prognostic values of the replication protein A family in gastric cancer.

The replication protein A (RPA)1-4 family are single-stranded DNA-binding proteins that are essential components of DNA replication, repair and recombination, and cell cycle regulation. The present study aimed to evaluate the prognostic value of the RPA family members in patients with gastric cancer (GC), using datasets retrieved from the Oncomine public database. Datasets were retrieved for the purpose of comparing the RPA expression levels between GC and normal tissues. Additionally, Kaplan-Meier analysis was used to compare the overall survival (OS) times of GC patients that expressed different levels of RPA proteins. RPA1, 2, and 3 expression levels were all significantly upregulated in gastric intestinal-type, diffuse gastric, and gastric mixed adenocarcinomas, compared with those in normal mucosal tissues. Moreover, high mRNA expression levels of RPA3 and 4 predicted poorer OS times in all GCs, as well as patients with human epidermal growth factor receptor 2-negative and -positive GC. The high-risk group, separated by RPA signature, showed a poorer outcome than the low-risk group. RPA3 was the most strongly correlated with CD4+ T-cell levels. In conclusion, RPAs are novel prognostic indicators in GC, and can also predict the features of immunological diseases. Future experimental investigation into the roles of RPAs concerning the pathogenesis and development of GC may provide a novel biomarker or therapeutic target, improving the prognosis of patients with GC.

Dual functions for the ssDNA-binding protein RPA in meiotic recombination.

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.

Replication Protein A 3 Is Associated with Hepatocellular Carcinoma Tumorigenesis and Poor Patient Survival

Background: Replication protein A (RPA) 3 is a subunit of the RPA protein complex, which functions in multiple processes of DNA metabolism. Dysregulation of RPA1 and RPA2 has been implicated in tumor progression in several cancer types. However, the function of RPA3 in hepatocellular carcinoma (HCC) tumorigenesis has not been elucidated. Method: In this study, we investigated the function of RPA3 in HCC development by stably knocking down its expression using short hairpin RNA (shRNA) in HepG2 cell line, followed by cell proliferation, colony formation, soft agar, and invasion assays. Xenograft experiment was performed to examine in vivo tumor-promoting properties of RPA3. Results: Downregulation of RPA3-inhibited cell proliferation, colony formation, soft agar growth as well as invasion in HepG2 cells were observed. Stable knockdown of RPA3 significantly inhibited tumor growth in the xenograft mouse model. In addition, qRT-PCR analysis revealed that RPA3 was upregulated in human HCC tissues compared with matched noncancerous adjacent tissues (NATs). High expression of RPA3 was associated with poor overall survival and disease-free survival. Conclusion: Elevated expression of RPA3 promotes tumor progression in HCC cells. RPA3 is upregulated in HCC tissues and high expression of RPA3 is associated with poorer patient survival. Therefore, this protein may represent a novel therapeutic target for intervention of HCC and prognostic biomarker for patient survival.

Abstract 1416: Development of small molecule inhibitors for cancer therapy by targeting RPA and XPA nucleotide excision repair proteins

Abstract Targeting DNA repair and the DNA damage response for cancer therapy has recently gained increasing attention with inhibitors of the PARP enzyme showing a therapeutic efficacy in various cancers. Solid tumors of the lung, pancreas, breast, and ovary represent a continuing clinical challenge in treatment and together account for over 250,000 deaths in the US alone, representing over 40% of all cancer deaths. There are limited therapeutic options for these patients, and targeted and combination therapies remain necessary for treating these aggressive cancers. The opportunity exists to exploit recent scientific advances in our knowledge of the underlying biology behind these cancers to create novel targeted therapeutics to dramatically enhance patient response to therapy and ultimately survival. To this end, we have developed a series of novel small chemical molecules that disrupt critical protein-DNA interactions in the nucleotide excision repair (NER) pathways. It is well understood that various cancer treatments like cisplatin, etoposide and ionizing radiation impart their chemotherapeutic effect by the formation of direct DNA damage which block DNA replication and transcription culminating in apoptosis. It is also well established that repair of this DNA damage by nucleotide excision repair (NER) or homologous recombination repair (HRR) reduces the effectiveness of chemo- or radio- therapy. Replication protein A (RPA) and Xeroderma Pigmentosum Group A (XPA) plays a crucial role in the NER pathway and makes them a novel drug target to develop novel cancer therapy. We anticipate both direct mechanisms of action on the repair pathways and synthetic lethal interactions can be exploited for therapeutic benefit. The series of novel small molecule inhibitors that we have developed targeting RPA and XPA proteins independently exhibit single-agent anti-cancer activity in cancer cell lines, and potentiate cellular sensitivity to chemotherapeutic agent. Data demonstrate that these novel inhibitors do not interact with DNA but directly bind the corresponding NER proteins. Our data demonstrate that this class of inhibitors can be further developed as an anti-cancer therapeutic with considerable potential to be used in conjunction with radiation therapy and other cancer therapies that induce DNA damage. This works was supported by NIH grants R01-CA180710 and R41-CA162648 and the Tom and Julie Wood Family Foundation. Citation Format: Navnath S. Gavande, Pamela S. VanderVere-Carozza, Tyler L. Vernon, Katherine Pawelczak, John J. Turchi. Development of small molecule inhibitors for cancer therapy by targeting RPA and XPA nucleotide excision repair proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2017-1416

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

CTF18 interacts with replication proteinA in response to replication stress.

Replication stress response is a protective mechanism against defects in chromosome replication for maintaining genome integrity in eukaryotic cells. An alternative clamp loader complex termed chromosome transmission fidelity protein 18 and replication factorC (CTF18‑RFC) has been shown to act as a positive regulator of two types of replication stress response: S‑phase checkpoint signaling and translesion DNA synthesis. However, it remains largely unknown how CTF18‑RFC responds to replication stress and is recruited to stalled replication forks. The present study demonstrated that endogenous CTF18 forms a physical complex with a single‑stranded DNA‑binding protein replication proteinA (RPA) in mammalian cells. Using an in situ proximity ligation assay (PLA), it was demonstrated that the interaction between CTF18 and RPA occurs in chromatin when replication stress is elicited by treatment with hydroxyurea during S phase. Similar results were obtained after exposure to ultraviolet irradiation, which triggers translesion DNA synthesis. Furthermore, the PLA demonstrated that the kinetics of the interaction between CTF18 and RPA was positively correlated with that of checkpoint kinase 1 phosphorylation, which is an indicator of activation of the ATM and Rad3‑related pathway. These findings provide novel insights into the molecular mechanism underlying the participation of CTF18‑RFC in the regulation of replication stress response.

SLFN11 inhibits checkpoint maintenance and homologous recombination repair.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

RPA inhibition increases replication stress and suppresses tumor growth.

The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions that rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress. Cancer Res; 74(18); 5165-72. ©2014 AACR.

Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

Replication protein A (RPA) is the main eukaryotic ssDNA-binding protein with essential roles in DNA replication, recombination, and repair. RPA maintains the DNA as single-stranded and also interacts with other DNA-processing proteins, coordinating their assembly and disassembly on DNA. RPA binds to ssDNA in two conformational states with opposing affinities for DNA and proteins. The RPA-protein interactions are compatible with a low DNA affinity state that involves DNA-binding domain A (DBD-A) and DBD-B but not with the high DNA affinity state that additionally engages DBD-C and DBD-D. The structure of the high-affinity RPA-ssDNA complex reported here shows a compact quaternary structure held together by a four-way interface between DBD-B, DBD-C, the intervening linker (BC linker), and ssDNA. The BC linker binds into the DNA-binding groove of DBD-B, mimicking DNA. The associated conformational change and partial occlusion of the DBD-A-DBA-B protein-protein interaction site establish a mechanism for the allosteric coupling of RPA-DNA and RPA-protein interactions.

Replication Protein A Unfolds G-Quadruplex Structures with Varying Degrees of Efficiency

Replication protein A (RPA) is known to interact with guanine- (G-) rich sequences that adopt G-quadruplex (GQ) structures. Most studies reported in the literature were performed on GQ formed by homogeneous sequences, such as the human telomeric repeat, and RPA's ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ-forming DNA sequences (PQSs) to their stability against RPA-mediated unfolding using single-molecule fluorescence resonance energy transfer (FRET) and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQSs in the genome. The three GQ constructs have thermal stabilities that differ significantly. Our measurements showed that the most thermally stable structure (Tm = 86 °C) was also the most stable against RPA-mediated unfolding, although the least thermally stable structure (Tm = 69 °C) had at least an order-of-magnitude higher stability against RPA-mediated unfolding than the structure with intermediate thermal stability (Tm = 78 °C). The significance of this observation becomes more evident when considered within the context of the cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these results, we conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single-molecule stopped-flow method. All three GQ structures were unfolded within Δt ≈ 0.30 ± 0.10 s, a surprising result considering that the unfolding time does not correlate with thermal stability or stability against RPA-mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.