Ignacio F. Gallardo

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Significance Exonuclease 1 (Exo1) is a conserved eukaryotic nuclease that participates in DNA repair and telomere maintenance. Here we use high-throughput single-molecule imaging to examine Exo1 activity on DNA and in the presence of single-stranded DNA binding proteins. We report that both human and yeast Exo1 are processive nucleases but are rapidly turned over by replication protein A (RPA). In the presence of RPA, Exo1 retains limited DNA-processing activity, albeit via a distributive binding mechanism. This rapid turnover by RPA can appear stimulatory or inhibitory in gel-based assays, clarifying conflicting results in the existing literature. RPA-depleted human cells show elevated Exo1 loading but reduced overall DNA resection, underscoring the many roles of RPA in regulating DNA resection in vivo. Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.

Apyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall Changes Characteristic of Stress Responses

Suppressing the expression of two apyrase genes raises extracellular ATP levels and induces gene expression, growth, and cell wall changes characteristic of stress responses, thus implicating extracellular nucleotides as early signals linking biotic and abiotic stresses to growth inhibition. Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.

Tethering hydrophobic peptides to functionalized self-assembled monolayers on gold through two chemical linkers using the Huisgen cycloaddition.

Gold surfaces functionalized with an α-helical peptide have been generated by reacting an azide-terminated self-assembled monolayer with structured peptides containing two cyanophenylalanines through a Huisgen cycloaddition. Mixed monolayers of a reactive bromine-terminated thiol and inert alkane thiol were prepared at various concentrations of the Br-terminated moiety. These were reacted with sodium azide to form azide-terminated monolayers with controlled concentration of the reactive azide. These surfaces were studied through ellipsometry and X-ray photoelectron spectroscopy, which demonstrated that the concentration of the reactive azide group on the surface is controlled by the chemical conditions under which the monolayer is prepared. Grazing incident angle surface infrared spectroscopy (GRAS-IR) of the azide-terminated surface demonstrated that the azide is approximately perpendicular to the plane of the surface, as expected. These surfaces were then exposed to an α-helical peptide composed of alternating leucine and lysine residues, with two residues replaced with cyanophenylalanine to react with two neighboring surface-bound azide groups to bind the peptide to the surface through two covalent bonds. The yield of this reaction was quantified through monitoring the absorption of the azide group by GRAS-IR. Despite damage to the monolayer during the reaction, reaction yields of 80-98% were determined for optimized reaction conditions. Although the peptide retains its α-helical configuration under the reaction conditions, GRAS-IR analysis of the amide I and II modes of the surface-bound peptide showed that it is probably randomly oriented on the surface.

Demonstration of α-helical structure of peptides tethered to gold surfaces using surface infrared and circular dichroic spectroscopies.

Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.

High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging

Single-molecule studies of protein-DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains-a method for organizing arrays of DNA molecules on a fluid lipid bilayer-has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900,000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3-a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.

Coordination of Rad1–Rad10 interactions with Msh2–Msh3, Saw1 and RPA is essential for functional 3′ non-homologous tail removal

Abstract Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3′ non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1–Rad10 is the structure-specific endonuclease that cleaves the tails in 3′ non-homologous tail removal (3′ NHTR). Rad1–Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1–Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2–Msh3 and Saw1 recruit Rad1–Rad10 in 3′ NHTR; Rad14 recruits Rad1–Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3′ NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3′ NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A–Rad10 to recombination intermediates is defective. Interactions among rad1R218A–Rad10 and Msh2–Msh3 and Saw1 are altered and rad1R218A–Rad10 interactions with RPA are compromised. We propose a model in which Rad1–Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1–Rad10 and Msh2–Msh3, Saw1 and Msh2–Msh3 and Rad1–Rad10 and RPA. When any of these interactions is altered, 3′ NHTR is impaired.

Distinct roles of XPF-ERCC1 and Rad1-Rad10-Saw1 in replication-coupled and uncoupled inter-strand crosslink repair

Yeast Rad1–Rad10 (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1–Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1–Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1–Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1–Rad10 removes ICL via NER, whereas in S/G2, Rad1–Rad10 facilitates NER-independent replication-coupled ICLR.The yeast Rad1–Rad10 complex has multiple roles in DNA damage repair. Here the authors uncover mutants that uncouple the roles in UV excision repair and non-NER functions.

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

Tuning the absorption and emission of CdSe and ZnS core-shell nanoparticles by laser radiation

CdSe and ZnS core-shell nanoparticles made by LAM (Laser Ablation of Microparticles) show photoluminesence (PL) peaks in a region of wavelengths around 400 nm. Control over the size and PL peak position is obtained by irradiating the nanoparticles multiple times. In LAM, micropaticle powder passes through an aerosol generator and then into a laser ablation glass cell, where a laser pulse (high energy excimer laser) ablates the microparticle aerosol. Nanoparticles are formed after condensation. At this stage the nanoparticles can be covered with a second material or irradiated multiple times to change their size. The size distribution of these particles is successfully investigated with TEM (Transmission Electron Microscopy). PL blue shifts are seen as the mean size decreases.