Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. The binding to and dissociation of the four individual DNA binding domains (DBDs) from DNA result in configurational dynamics of the RPA-DNA complexes which are essential for replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM), mass photometry (MP) with biophysical and biochemical analyses, of a gain-of-function RPA mutant to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a stable ternary complex. Uniquely among hnRNPA1 target RNAs, TERRA is capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by TERRA RNA.

Single-molecule analysis of the improved variants of the G-quadruplex recognition protein G4P.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al . synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro , and to display better selectivity towards G4s than the previously published BG4 antibody. To get insight into the G4P-G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 µM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 µM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase ß during base excision repair.

Base excision repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase ß (Pol ß). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol ß substrate) were subsequently bound by Pol ß, the Pol ß enzyme dissociated shortly after binding in most of the observations. In the events where Pol ß binding was followed by APE1 dissociation during substrate channeling, Pol ß remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol ß during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5' overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein.

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.

Making Choices: DNA Replication Fork Recovery Mechanisms.

DNA replication is laden with obstacles that slow, stall, collapse, and break DNA replication forks. At each obstacle, there is a decision to be made whether to bypass the lesion, repair or restart the damaged fork, or to protect stalled forks from further demise. Each "decision" draws upon multitude of proteins participating in various mechanisms that allow repair and restart of replication forks. Specific functions for many of these proteins have been described and an understanding of how they come together in supporting replication forks is starting to emerge. Many questions, however, remain regarding selection of the mechanisms that enable faithful genome duplication and how "normal" intermediates in these mechanisms are sometimes funneled into "rogue" processes that destabilize the genome and lead to cancer, cell death, and emergence of chemotherapeutic resistance. In this review we will discuss molecular mechanisms of DNA damage bypass and replication fork protection and repair. We will specifically focus on the key players that define which mechanism is employed including: PCNA and its control by posttranslational modifications, translesion synthesis DNA polymerases, molecular motors that catalyze reversal of stalled replication forks, proteins that antagonize fork reversal and protect reversed forks from nucleolytic degradation, and the machinery of homologous recombination that helps to reestablish broken forks. We will also discuss risks to genome integrity inherent in each of these mechanisms.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.

Switch-like control of helicase processivity by single-stranded DNA binding protein.

Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. Ferroplasma acidarmanus xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent 'processivity switch' in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis.

Replication Protein A (RPA) is a critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determined that RPA-2 is critical for germline replication and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for somatic replication, in contrast to other organisms that require both subunits. Six different hetero- and homodimeric complexes containing permutations of RPA-1, RPA-2 and RPA-4 can be detected in whole animal extracts. Our in vivo studies indicate that RPA-1/4 dimer is less abundant in the nucleus and its formation is inhibited by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibits RAD-51 filament formation and promotes apoptosis of a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans.

Reversible Al Propagation in Si x Ge1-x Nanowires: Implications for Electrical Contact Formation.

While reversibility is a fundamental concept in thermodynamics, most reactions are not readily reversible, especially in solid-state physics. For example, thermal diffusion is a widely known concept, used among others to inject dopants into the substitutional positions in the matrix and improve device properties. Typically, such a diffusion process will create a concentration gradient extending over increasingly large regions, without possibility to reverse this effect. On the other hand, while the bottom-up growth of semiconducting nanowires is interesting, it can still be difficult to fabricate axial heterostructures with high control. In this paper, we report a thermally assisted partially reversible thermal diffusion process occurring in the solid-state reaction between an Al metal pad and a Si x Ge1-x alloy nanowire observed by in situ transmission electron microscopy. The thermally assisted reaction results in the creation of a Si-rich region sandwiched between the reacted Al and unreacted Si x Ge1-x part, forming an axial Al/Si/Si x Ge1-x heterostructure. Upon heating or (slow) cooling, the Al metal can repeatably move in and out of the Si x Ge1-x alloy nanowire while maintaining the rodlike geometry and crystallinity, allowing to fabricate and contact nanowire heterostructures in a reversible way in a single process step, compatible with current Si-based technology. This interesting system is promising for various applications, such as phase change memories in an all crystalline system with integrated contacts as well as Si/Si x Ge1-x /Si heterostructures for near-infrared sensing applications.

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

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.

Correlated and in-situ electrical transmission electron microscopy studies and related membrane-chip fabrication.

Understanding the interplay between the structure, composition and opto-electronic properties of semiconductor nano-objects requires combining transmission electron microscopy (TEM) based techniques with electrical and optical measurements on the very same specimen. Recent developments in TEM technologies allow not only the identification and in-situ electrical characterization of a particular object, but also the direct visualization of its modification in-situ by techniques such as Joule heating. Over the past years, we have carried out a number of studies in these fields that are reviewed in this contribution. In particular, we discuss here i) correlated studies where the same unique object is characterized electro-optically and by TEM, ii) in-situ Joule heating studies where a solid-state metal-semiconductor reaction is monitored in the TEM, and iii) in-situ biasing studies to better understand the electrical properties of contacted single nanowires. In addition, we provide detailed fabrication steps for the silicon nitride membrane-chips crucial to these correlated and in-situ measurements.

Physiological and Pathological Roles of RAD52 at DNA Replication Forks.

Understanding basic molecular mechanisms underlying the biology of cancer cells is of outmost importance for identification of novel therapeutic targets and biomarkers for patient stratification and better therapy selection. One of these mechanisms, the response to replication stress, fuels cancer genomic instability. It is also an Achille's heel of cancer. Thus, identification of pathways used by the cancer cells to respond to replication-stress may assist in the identification of new biomarkers and discovery of new therapeutic targets. Alternative mechanisms that act at perturbed DNA replication forks and involve fork degradation by nucleases emerged as crucial for sensitivity of cancer cells to chemotherapeutics agents inducing replication stress. Despite its important role in homologous recombination and recombinational repair of DNA double strand breaks in lower eukaryotes, RAD52 protein has been considered dispensable in human cells and the full range of its cellular functions remained unclear. Very recently, however, human RAD52 emerged as an important player in multiple aspects of replication fork metabolism under physiological and pathological conditions. In this review, we describe recent advances on RAD52's key functions at stalled or collapsed DNA replication forks, in particular, the unexpected role of RAD52 as a gatekeeper, which prevents unscheduled processing of DNA. Last, we will discuss how these functions can be exploited using specific inhibitors in targeted therapy or for an informed therapy selection.