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.

New insights into DNA polymerase mechanisms provided by time-lapse crystallography.

DNA polymerases play central roles in DNA replication and repair by catalyzing template-directed nucleotide incorporation. Recently time-lapse X-ray crystallography, which allows one to observe reaction intermediates, has revealed numerous and unexpected mechanistic features of DNA polymerases. In this article, we will examine recent new discoveries that have come from time-lapse crystallography that are currently transforming our understanding of the structural mechanisms used by DNA polymerases. Among these new discoveries are the binding of a third metal ion within the polymerase active site, the mechanisms of translocation along the DNA, the presence of new fidelity checkpoints, a novel pyrophosphatase activity within the active site, and the mechanisms of pyrophosphorolysis.

Recent Advances in Understanding the Structures of Translesion Synthesis DNA Polymerases.

DNA damage in the template strand causes replication forks to stall because replicative DNA polymerases are unable to efficiently incorporate nucleotides opposite template DNA lesions. To overcome these replication blocks, cells are equipped with multiple translesion synthesis polymerases that have evolved specifically to incorporate nucleotides opposite DNA lesions. Over the past two decades, X-ray crystallography has provided a wealth of information about the structures and mechanisms of translesion synthesis polymerases. This approach, however, has been limited to ground state structures of these polymerases bound to DNA and nucleotide substrates. Three recent methodological developments have extended our understanding of the structures and mechanisms of these polymerases. These include time-lapse X-ray crystallography, which allows one to identify novel reaction intermediates; full-ensemble hybrid methods, which allow one to examine the conformational flexibility of the intrinsically disordered regions of proteins; and cryo-electron microscopy, which allows one to determine the high-resolution structures of larger protein complexes. In this article, we will discuss how these three methodological developments have added to our understanding of the structures and mechanisms of translesion synthesis polymerases.

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.

Visualizing Rev1 catalyze protein-template DNA synthesis.

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

Yeast DNA polymerase η possesses two PIP-like motifs that bind PCNA and Rad6-Rad18 with different specificities.

In translesion synthesis (TLS), specialized DNA polymerases, such as polymerase (pol) η and Rev1, are recruited to stalled replication forks. These polymerases form a multi-protein complex with PCNA, Rad6-Rad18, and other specialized polymerases. Pol η interacts with PCNA and Rev1 via a PCNA-interacting protein (PIP) motif in its C-terminal unstructured region. Here we report the discovery of a second PIP-like motif in the C-terminal region of pol η, which we have designated as PIP2. We have designated the original PIP motif as PIP1. We show that the pol η PIP1 and PIP2 motifs bind PCNA with different affinities and kinetics. PIP1 binds with higher affinity than does PIP2, and PIP1 dissociates more slowly than does PIP2. In addition, we show that the interaction between pol η and Rad6-Rad18 is also mediated by the pol η PIP1 and PIP2 motifs. Again, we show that the affinity and kinetics by which these motifs bind Rad6-Rad18 is different. These findings are significant, because the multiple PIP-like motifs on pol η likely play quite different roles within the multi-protein complex formed at stalled replication forks. PIP1 likely plays a critical role in the recruiting pol η to this multi-protein complex. PIP2, by contrast, likely plays a critical role in maintaining the architecture and the dynamics of this multi-protein complex needed to maximize the efficiency and accuracy of TLS.

Control of DNA Damage Bypass by Ubiquitylation of PCNA.

DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.

Conformational flexibility of fork-remodeling helicase Rad5 shown by full-ensemble hybrid methods.

Several pathways exist to bypass DNA damage during replication. One such pathway is template switching. The Rad5 protein plays two important roles in template switching: it is an E3 ubiquitin ligase that catalyzes PCNA poly-ubiquitylation and it is a helicase that converts replication forks to chicken foot structures. To understand the structure, conformational flexibility, and mechanism of Rad5, we used a full-ensemble hybrid method combining Langevin dynamics simulations and small-angle X-ray scattering. From these studies, we generated the first experimentally validated, high-resolution structural model of Rad5. We found that Rad5 is more compact and less extended than is suggested by its large amount of predicted intrinsic disorder. Thus, Rad5 likely has a novel intra-molecular interaction that limits the range of conformational space it can sample. We provide evidence for a novel interaction between the HIRAN and the helicase domains of Rad5, and we discuss the biological and mechanistic implications of this.

Modeling Conformationally Flexible Proteins With X-ray Scattering and Molecular Simulations.

Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.

Conformational Flexibility of Ubiquitin-Modified and SUMO-Modified PCNA Shown by Full-Ensemble Hybrid Methods.

Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.

Eukaryotic translesion synthesis: Choosing the right tool for the job.

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases are thought to form a complex at the stalled replication fork that we refer to as the mutasome. This hypothetical multi-protein complex is structurally organized by the replication accessory factor PCNA and the non-classical polymerase Rev1. One of the non-classical polymerases within this complex then catalyzes replication through the damage. Each non-classical polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical polymerase is chosen to bypass the damage. In this review article, we discuss how the most appropriate polymerase is chosen. In so doing, we examine the structural motifs that mediate the protein interactions in the mutasome; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome.

Crystal structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction site on the front face of the PCNA ring.

Proliferating cell nuclear antigen (PCNA), a homotrimeric protein, is the eukaryotic sliding clamp that functions as a processivity factor for polymerases during DNA replication. Chromatin association factor 1 (CAF-1) is a heterotrimeric histone chaperone protein that is required for coupling chromatin assembly with DNA replication in eukaryotes. CAF-1 association with replicating DNA, and the targeting of newly synthesized histones to sites of DNA replication and repair requires its interaction with PCNA. Genetic studies have identified three mutant forms of PCNA in yeast that cause defects in gene silencing and exhibit altered association of CAF-1 to chromatin in vivo, as well as inhibit binding to CAF-1 in vitro. Three of these mutant forms of PCNA, encoded by the pol30-6, pol30-8, and the pol30-79 alleles, direct the synthesis of PCNA proteins with the amino acid substitutions D41A/D42A, R61A/D63A, and L126A/I128A, respectively. Interestingly, these double alanine substitutions are located far away from each other within the PCNA protein. To understand the structural basis of the interaction between PCNA and CAF-1 and how disruption of this interaction leads to reduced gene silencing, we determined the X-ray crystal structures of each of these mutant PCNA proteins. All three of the substitutions caused disruptions of a surface cavity on the front face of the PCNA ring, which is formed in part by three loops comprised of residues 21-24, 41-44, and 118-134. We suggest that this cavity is a novel binding pocket required for the interaction between PCNA and CAF-1, and that this region in PCNA also represents a potential binding site for other PCNA-binding proteins.

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility.

Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches-including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering-we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.

Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases.

Translesion synthesis is the process by which nonclassical DNA polymerases bypass DNA damage during DNA replication. Cells possess a variety of nonclassical polymerases, each one is specific for incorporating nucleotides opposite to one or more closely related DNA lesions, called its cognate lesions. In this chapter, we discuss a variety of approaches for probing the catalytic activities and the protein-protein interactions of nonclassical polymerases. With respect to their catalytic activities, we discuss polymerase assays, steady-state kinetics, and presteady-state kinetics. With respect to their interactions, we discuss qualitative binding assays such as enzyme-linked immunosorbent assays and coimmunoprecipitation; quantitative binding assays such as isothermal titration calorimetry, surface plasmon resonance, and nuclear magnetic resonance spectroscopy; and single-molecule binding assays such as total internal reflection fluorescence microscopy. We focus on how nonclassical polymerases accommodate their cognate lesions during nucleotide incorporation and how the most appropriate nonclassical polymerase is selected for bypassing a given lesion.

Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases.

DNA replication is constantly challenged by DNA lesions, noncanonical DNA structures and difficult-to-replicate DNA sequences. Two major strategies to rescue a stalled replication fork and to ensure continuous DNA synthesis are: (1) template switching and recombination-dependent DNA synthesis; and (2) translesion synthesis (TLS) using specialized DNA polymerases to perform nucleotide incorporation opposite DNA lesions. The former pathway is mainly error-free, and the latter is error-prone and a major source of mutagenesis. An accepted model of translesion synthesis involves DNA polymerase switching steps between a replicative DNA polymerase and one or more TLS DNA polymerases. The mechanisms that govern the selection and exchange of specialized DNA polymerases for a given DNA lesion are not well understood. In this review, recent studies concerning the mechanisms of selection and switching of DNA polymerases in eukaryotic systems are summarized.

DNA Polymerase Fidelity: Beyond Right and Wrong.

Accurate DNA replication depends on the ability of DNA polymerases to discriminate between correctly and incorrectly paired nucleotides. In this issue of Structure, Batra et al. (2016) show the structural basis for why DNA polymerases do not efficiently add correctly paired nucleotides immediately after incorporating incorrectly paired ones.

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today.