Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities.

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.

Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single-molecule studies.

SignificanceWe directly visualize DNA translocation and lesion recognition by the O6-alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn2+ occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.

Direct hOGG1-Myc interactions inhibit hOGG1 catalytic activity and recruit Myc to its promoters under oxidative stress.

The base excision repair (BER) glycosylase hOGG1 (human oxoguanine glycosylase 1) is responsible for repairing oxidative lesions in the genome, in particular oxidised guanine bases (oxoG). In addition, a role of hOGG1 in transcription regulation by recruitment of various transcription factors has been reported. Here, we demonstrate direct interactions between hOGG1 and the medically important oncogene transcription factor Myc that is involved in transcription initiation of a large number of genes including inflammatory genes. Using single molecule atomic force microscopy (AFM), we reveal recruitment of Myc to its E-box promoter recognition sequence by hOGG1 specifically under oxidative stress conditions, and conformational changes in hOGG1-Myc complexes at oxoG lesions that suggest loading of Myc at oxoG lesions by hOGG1. Importantly, our data show suppression of hOGG1 catalytic activity in oxoG repair by Myc. Furthermore, mutational analyses implicate the C28 residue in hOGG1 in oxidation induced protein dimerisation and suggest a role of hOGG1 dimerisation under oxidising conditions in hOGG1-Myc interactions. From our data we develop a mechanistic model for Myc recruitment by hOGG1 under oxidising, inflammatory conditions, which may be responsible for the observed enhanced gene expression of Myc target genes.

Recent Advances in Genome Maintenance Processes.

Given life's dependence on genome maintenance, unsurprisingly, investigations of the molecular processes involved in protecting the genome or, failing this, repairing damages to and alterations introduced into genetic material are at the forefront of current research [...].

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase.

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.

Alkyltransferase-like protein clusters scan DNA rapidly over long distances and recruit NER to alkyl-DNA lesions.

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.

The DNA Alkyltransferase Family of DNA Repair Proteins: Common Mechanisms, Diverse Functions.

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O6-alkylguanine and O4-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O6-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.

SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication.

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

Engineering Nanogels for Drug Delivery to Pathogenic Fungi Aspergillus fumigatus by Tuning Polymer Amphiphilicity.

Invasive aspergillosis is a serious threat to immunodeficient and critically ill patients caused mainly by the fungus Aspergillus fumigatus. Here, poly(glycidol)-based nanogels (NGs) are proposed as delivery vehicles for antifungal agents for sustained drug release. NGs are formed by simple self-assembly of random copolymers, followed by oxidative cross-linking of thiol functionalities. We investigate the impact of copolymer amphiphilicity on NG interaction with mature fungal hyphae in order to select the optimal drug delivery system for model antifungal drug amphotericin B. The results show that drug-loaded NGs decrease minimal inhibitory concentration (MIC) for around four times and slow down the fungal biofilm synthesis at concentrations lower than MIC. Our results suggest that amphiphilicity of nanoparticle's polymer matrix is an important factor in understanding the action of nanocarriers toward fungal cells and should be considered in the development of nanoparticle-based antifungal therapy.

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Cdc45-induced loading of human RPA onto single-stranded DNA.

Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork.

A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition.

Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes.

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging.

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.

Strand-specific recognition of DNA damages by XPD provides insights into nucleotide excision repair substrate versatility.

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Chronophin dimerization is required for proper positioning of its substrate specificity loop.

Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.

Cooperative cluster formation, DNA bending and base-flipping by O6-alkylguanine-DNA alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee-von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (∼0, ∼30 and ∼60°) that are consistent with models in which each protein induces a bend of ∼30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.

Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states.

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAA receptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

DNA substrate preparation for atomic force microscopy studies of protein-DNA interactions.

Protein-DNA interactions provide fundamental control mechanisms over biologically essential processes such as DNA replication, transcription, and repair. However, many details of these mechanisms still remain unclear. Atomic force microscopy (AFM) analyses provide unique and important structural and functional information on such protein-DNA interactions at the level of the individual molecules. The high sensitivity of the method with topographical visualization of all sample components also demands for extremely clean and pure materials. Here, we provide an overview of molecular biology-based approaches to produce DNA substrates for AFM imaging as well as other types of experiments, such as optical or magnetic tweezers, that profit from controllable substrate properties in long DNA fragments. We present detailed strategies to produce different types of motifs in DNA that are frequently employed targets of protein interactions. Importantly, the presented preparation techniques imply exact knowledge of the location of the introduced specific target sites within the DNA fragments, allowing for a distinction between specific and non-specific protein-DNA interactions in the AFM images and for separate conformational analyses of the different types of protein-DNA complexes.