Daniel Barsky

The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA

DNA continuously suffers the loss of its constituent bases, and thereby, a loss of potentially vital genetic information. Sites of missing bases--termed abasic or apurinic/apyrimidinic (AP) sites--form spontaneously, through damage-induced hydrolytic base release, or by enzyme-catalyzed removal of modified or mismatched bases during base excision repair (BER). In this review, we discuss the structural and biological consequences of abasic lesions in DNA, as well as the multiple repair pathways for such damage, while emphasizing the mechanistic operation of the multi-functional human abasic endonuclease APE1 (or REF-1) and its potential relationship to disease.

Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.

Integrating Ion Mobility Mass Spectrometry with Molecular Modelling to Determine the Architecture of Multiprotein Complexes

Current challenges in the field of structural genomics point to the need for new tools and technologies for obtaining structures of macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) and incomplete atomic structures, usually from X-ray crystallography, to generate models of the subunit architecture of protein complexes. We begin by analyzing protein complexes using IM-MS, and by taking measurements of both intact complexes and sub-complexes that are generated in solution. We then examine available high resolution structural data and use a suite of computational methods to account for missing residues at the subunit and/or domain level. High-order complexes and sub-complexes are then constructed that conform to distance and connectivity constraints imposed by IM-MS data. We illustrate our method by applying it to multimeric protein complexes within the Escherichia coli replisome: the sliding clamp, (β2), the γ complex (γ3δδ′), the DnaB helicase (DnaB6) and the Single-Stranded Binding Protein (SSB4).

Ion Mobility-Mass Spectrometry Reveals the Influence of Subunit Packing and Charge on the Dissociation of Multiprotein Complexes

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.

Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3′ end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

AS2TS system for protein structure modeling and analysis

We present a set of programs and a website designed to facilitate protein structure comparison and protein structure modeling efforts. Our protein structure analysis and comparison services use the LGA (local-global alignment) program to search for regions of local similarity and to evaluate the level of structural similarity between compared protein structures. To facilitate the homology-based protein structure modeling process, our AL2TS service translates given sequence–structure alignment data into the standard Protein Data Bank (PDB) atom records (coordinates). For a given sequence of amino acids, the AS2TS (amino acid sequence to tertiary structure) system calculates (e.g. using PSI-BLAST PDB analysis) a list of the closest proteins from the PDB, and then a set of draft 3D models is automatically created. Web services are available at .

Two processivity clamp interactions differentially alter the dual activities of UmuC

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV‐induced and chemically induced mutagenesis, possesses a canonical β processivity clamp‐binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV‐induced mutagenesis. Increased levels of wild‐type β can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold‐sensitive and β‐dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in β, we restored UV‐induced mutagenesis by a UmuC β‐binding motif variant. A recent co‐crystal structure of the ‘little finger’ domain of E. coli pol IV (DinB) with β suggests that, in addition to the canonical β‐binding motif, a second site of pol IV (303VWP305) interacts with β at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold‐sensitive phenotype. These two β interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the β clamp.

A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

UmuD2 cleaves and removes its N-terminal 24 amino acids to form UmuD′2, which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the β processivity clamp (“β”) of the replicative DNA polymerase, pol III. A possible β-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with β. We performed alanine-scanning mutagenesis of this motif (14TFPLF18) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the β-binding motif variant bind to β with similar Kd values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in β is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD′, such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD′ mimic.

The Mechanical Properties of PCNA: Implications for the Loading and Function of a DNA Sliding Clamp

Sliding clamps are toroidal proteins that encircle DNA and act as mobile platforms for DNA replication and repair machinery. To be loaded onto DNA, the eukaryotic sliding clamp Proliferating Cell Nuclear Antigen (PCNA) must be splayed open at one of the subunit-subunit interfaces by the ATP-dependent clamp loader, Replication Factor C, whose clamp-interacting sites form a right-handed spiral. Earlier molecular dynamics (MD) studies suggested that when PCNA opens, it preferentially adopts a right-handed spiral to match the spiral of the clamp loader. Here, analysis of considerably longer MD simulations shows that although the opened form of PCNA can achieve conformations matching the helical pitch of Replication Factor C, it is not biased toward a right-handed spiral structure. A coarse-grained elastic model was also built; its strong correspondence to the all-atom MD simulations of PCNA suggests that the behavior of the open clamp is primarily due to elastic deformation governed by the topology of the clamp domains. The elastic model was further used to construct the energy landscape of the opened PCNA clamp, including conformations that would allow binding to the clamp loader and loading onto double-stranded DNA. A picture of PCNA emerges of a rather flexible protein that, once opened, is mechanically compliant in the clamp opening process.