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.

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.

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.

R.I.P. to the PIP: PCNA-binding motif no longer considered specific: PIP motifs and other related sequences are not distinct entities and can bind multiple proteins involved in genome maintenance.

Many proteins responsible for genome maintenance interact with one another via short sequence motifs. The best known of these are PIP motifs, which mediate interactions with the replication protein PCNA. Others include RIR motifs, which bind the translesion synthesis protein Rev1, and MIP motifs, which bind the mismatch repair protein Mlh1. Although these motifs have similar consensus sequences, they have traditionally been viewed as separate motifs, each with their own target protein. In this article, we review several recent studies that challenge this view. Taken together, they imply that these different motifs are not distinct entities. Instead, there is a single, broader class of motifs, which we call "PIP-like" motifs, which have overlapping specificities and are capable of binding multiple target proteins. Given this, we must reassess the role of these motifs in forming the network of interacting proteins responsible for genome maintenance.

PCNA tool belts and polymerase bridges form during translesion synthesis.

Large multi-protein complexes play important roles in many biological processes, including DNA replication and repair, transcription, and signal transduction. One of the challenges in studying such complexes is to understand their mechanisms of assembly and disassembly and their architectures. Using single-molecule total internal reflection (TIRF) microscopy, we have examined the assembly and disassembly of the multi-protein complex that carries out translesion synthesis, the error-prone replication of damaged DNA. We show that the ternary complexes containing proliferating cell nuclear antigen (PCNA) and two non-classical DNA polymerases, Rev1 and DNA polymerase η, have two architectures: PCNA tool belts and Rev1 bridges. Moreover, these complexes are dynamic and their architectures can interconvert without dissociation. The formation of PCNA tool belts and Rev1 bridges and the ability of these complexes to change architectures are likely means of facilitating selection of the appropriate non-classical polymerase and polymerase-switching events.

The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1.

Y-family DNA polymerases, such as polymerase η, polymerase ι, and polymerase κ, catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase η, a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase η mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase η binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase κ and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed.

Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures.

A balance of van der Waals, electrostatic, and hydrophobic forces drive the folding and packing of protein side chains. Although such interactions between residues are often approximated as being pairwise additive, in reality, higher-order many-body contributions that depend on environment drive hydrophobic collapse and cooperative electrostatics. Beginning from dead-end elimination, we derive the first algorithm, to our knowledge, capable of deterministic global repacking of side chains compatible with many-body energy functions. The approach is applied to seven PCNA x-ray crystallographic data sets with resolutions 2.5-3.8 Å (mean 3.0 Å) using an open-source software. While PDB_REDO models average an Rfree value of 29.5% and MOLPROBITY score of 2.71 Å (77th percentile), dead-end elimination with the polarizable AMOEBA force field lowered Rfree by 2.8-26.7% and improved mean MOLPROBITY score to atomic resolution at 1.25 Å (100th percentile). For structural biology applications that depend on side-chain repacking, including x-ray refinement, homology modeling, and protein design, the accuracy limitations of pairwise additivity can now be eliminated via polarizable or quantum mechanical potentials.

Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA.

PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)--a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations.

Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1.

Translesion synthesis (TLS) is a pathway in which specialized, low-fidelity DNA polymerases are used to overcome replication blocks caused by DNA damage. The use of this pathway often results in somatic mutations that can drive carcinogenesis. Rev1 is a TLS polymerase found in all eukaryotes that plays a pivotal role in mediating DNA damage-induced mutagenesis. It possesses a BRCA1 C-terminal (BRCT) domain that is required for its function. The rev1-1 allele encodes a mutant form of Rev1 with a G193R substitution in this domain, which reduces the level of DNA damage-induced mutagenesis. Despite its clear importance in mutagenic TLS, the role of the BRCT domain is unknown. Here, we report the X-ray crystal structure of the yeast Rev1 BRCT domain and show that substitutions in residues constituting its phosphate-binding pocket do not affect mutagenic TLS. This suggests that the role of the Rev1 BRCT domain is not to recognize phosphate groups on protein binding partners or on DNA. We also found that residue G193 is located in a conserved turn region of the BRCT domain, and our in vivo and in vitro studies suggest that the G193R substitution may disrupt Rev1 function by destabilizing the fold of the BRCT domain.

Distinct structural alterations in proliferating cell nuclear antigen block DNA mismatch repair.

During DNA replication, mismatches and small loops in the DNA resulting from insertions or deletions are repaired by the mismatch repair (MMR) machinery. Proliferating cell nuclear antigen (PCNA) plays an important role in both mismatch-recognition and resynthesis stages of MMR. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little, if any, other defects. The C22Y mutant PCNA protein completely blocks MutSα-dependent MMR, and the C81R mutant PCNA protein partially blocks both MutSα-dependent and MutSß-dependent MMR. In order to understand the structural and mechanistic basis by which these two amino acid substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. We found that these amino acid substitutions lead to subtle, distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in an extended loop near the PCNA subunit interface. We conclude that the structural integrity of the α-helices lining the central hole and this loop are both necessary to form productive complexes with MutSα and mismatch-containing DNA.

PCNA structure and function: insights from structures of PCNA complexes and post-translationally modified PCNA.

Proliferating cell nuclear antigen (PCNA), the eukaryotic DNA sliding clamp, forms a ring-shaped homo-trimer that encircles double-stranded DNA. This protein is best known for its ability to confer high processivity to replicative DNA polymerases. However, it does far more than this, because it forms a mobile platform on the DNA that recruits many of the proteins involved in DNA replication, repair, and recombination to replication forks. X-ray crystal structures of PCNA bound to PCNA-binding proteins have provided insights into how PCNA recognizes its binding partners and recruits them to replication forks. More recently, X-ray crystal structures of ubiquitin-modified and SUMO-modified PCNA have provided insights into how these post-translational modifications alter the specificity of PCNA for some of its binding partners. This article focuses on the insights gained from structural studies of PCNA complexes and post-translationally modified PCNA.

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.

Variations on a theme: eukaryotic Y-family DNA polymerases.

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase delta.

Eukaryotic DNA polymerase delta (pol delta) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol delta is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol delta is one of the major eukaryotic replicative polymerase, the mechanism by which it incorporates nucleotides is unknown. Here we report both steady state and pre-steady state kinetic studies of the fidelity of pol delta. We found that pol delta incorporates nucleotides with an error frequency of 10(-4) to 10(-5). Furthermore, we showed that for correct versus incorrect nucleotide incorporation, there are significant differences between both pre-steady state kinetic parameters (apparent K(d)(dNTP) and k(pol)). Somewhat surprisingly, we found that pol delta synthesizes DNA at a slow rate with a k(pol) of approximately 1 s(-1). We suggest that, unlike its prokaryotic counterparts, pol delta requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.

Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta.

DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.

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.