Visualizing the Path of DNA through Proteins Using DREEM Imaging.

Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many existing commercial AFMs, making the technique readily available.

Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA.

Fluorescently labeled oligonucleotides are powerful tools for characterizing DNA processes; however, their use is limited by the cost and sequence requirements of current labeling technologies. Here, we develop an easy, inexpensive, and sequence-independent method for site-specifically labeling DNA oligonucleotides. We utilize commercially synthesized oligonucleotides containing phosphorothioate diester(s) in which a nonbridging oxygen is replaced with a sulfur (PS-DNA). The increased nucleophilicity of the thiophosphoryl sulfur relative to the phosphoryl oxygen permits selective reactivity with iodoacetamide compounds. As such, we leverage a long-existing bifunctional linker, N,N'-bis(α-iodoacetyl)-2-2'-dithiobis(ethylamine) (BIDBE), that reacts with PS-DNAs to leave a free thiol, allowing conjugation of the wide variety of commercial maleimide-functionalized compounds. We optimized BIDBE synthesis and its attachment to PS-DNA and then fluorescently labeled the BIDBE-PS-DNA using standard protocols for labeling cysteines. We purified the individual epimers, and using single-molecule Förster resonance energy transfer (FRET), we show that the FRET efficiency is independent of the epimeric attachment. Subsequently, we demonstrate that an epimeric mixture of double-labeled Holliday junctions (HJs) can be used to characterize their conformational properties in the absence and presence of the structure-specific endonuclease Drosophila melanogaster Gen. Finally, we use a biochemical activity assay to show that this double-labeled HJ is functional for cleavage by Gen and that the double-labeled HJ allows multiple DNA species to be identified in a single experiment. In conclusion, our results indicate that dye-labeled BIDBE-PS-DNAs are comparable to commercially labeled DNAs at a significantly reduced cost. Notably, this technology could be applied to other maleimide-functionalized compounds, such as spin labels, biotin, and proteins. The sequence independence of labeling, coupled with its ease and low cost, enables unrestricted exploration of dye placement and choice, providing the potential for creation of differentially labeled DNA libraries and opening previously inaccessible experimental avenues.

Eukaryotic Mismatch Repair in Relation to DNA Replication.

Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome.

Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby.

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch.

Dynamic human MutSα-MutLα complexes compact mismatched DNA.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

CUREing biochemistry lab monotony.

Undergraduate research experience is critical to success in post-graduate research settings. The recent movement away from "cookbook" style labs to course-based undergraduate research experiences (CUREs) in undergraduate laboratories has allowed universities to provide inclusive research experience while bypassing the limitations of extracurricular apprenticeships. This paper describes an upper-level biochemistry CURE designed to provide students with an introductory experience to graduate-level research by studying a suspected DNA helicase. This CURE is designed to span multiple semesters, where each student cohort builds upon the work of previous semesters. Pre- and post-course surveys were employed to assess student confidence in bench skills, perceptions of the course, and project ownership. The results show that the incorporation of lab meeting-style recitations and poster presentations led to higher project ownership, while overcoming troubleshooting was a significant challenge. Furthermore, confidence in every experimental technique increased significantly in all but one instance. Based on these results, this CURE is providing students with a realistic experience in graduate-level research.

Ctp1 protein-DNA filaments promote DNA bridging and DNA double-strand break repair.

The Ctp1 protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination. Fission yeast Ctp1 and its budding yeast (Sae2) and human (CtIP) homologs control Mre11-Rad50-Nbs1 nuclease complex activity and harbor DNA-binding and -bridging activities. However, the molecular basis for Ctp1-DNA transactions remains undefined. Here, we report atomic force microscopy (AFM) imaging of S. pombe Ctp1-DNA complexes revealing that Ctp1 polymerizes on dsDNA molecules and forms synaptic filaments that bridge two dsDNA strands. We observed that Ctp1 DNA filaments are typified by an average filament length of ∼180 bp of dsDNA and a Ctp1 tetramer footprint of ∼15 bp. Biochemical results characterizing Ctp1 variants with impaired DNA-binding or -bridging properties were consistent with Ctp1-mediated DNA bridging requiring the intact and correctly folded Ctp1 tetramer. Furthermore, mutations altering Ctp1 oligomerization and DNA bridging in vitro conferred cell sensitivity to DSB-producing agents. Together, these results support an important role for Ctp1-regulated DNA strand coordination required for DNA DSB repair in S. pombe.

Fabrication of a Biocompatible Mica/Gold Surface for Tip-Enhanced Raman Spectroscopy.

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.

Coordinated protein and DNA conformational changes govern mismatch repair initiation by MutS.

MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA. Previous single-molecule FRET (smFRET) studies characterized the dynamics of MutS DNA-binding domains during these transitions. Here, we use protein-DNA and DNA-DNA smFRET to monitor DNA conformational changes, and we use kinetic analyses to correlate DNA and protein conformational changes to one another and to the steps on the pathway to mobile clamp formation. The results reveal multiple sequential structural changes in both MutS and DNA, and they suggest that DNA dynamics play a critical role in the formation of the MutS mobile clamp. Taking these findings together with data from our previous studies, we propose a unified model of coordinated MutS and DNA conformational changes wherein initiation of mismatch repair is governed by a balance of DNA bending/unbending energetics and MutS conformational changes coupled to its nucleotide binding properties.

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

We FRET so You Don't Have To: New Models of the Lipoprotein Lipase Dimer.

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL-LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.

MutL traps MutS at a DNA mismatch.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (ß-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by ß-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with ß-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling.

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.

Dynamics of MutS-mismatched DNA complexes are predictive of their repair phenotypes.

MutS recognizes base-base mismatches and base insertions/deletions (IDLs) in newly replicated DNA. Specific interactions between MutS and these errors trigger a cascade of protein-protein interactions that ultimately lead to their repair. The inability to explain why different DNA errors are repaired with widely varying efficiencies in vivo remains an outstanding example of our limited knowledge of this process. Here, we present single-molecule Förster resonance energy transfer measurements of the DNA bending dynamics induced by Thermus aquaticus MutS and the E41A mutant of MutS, which is known to have error specific deficiencies in signaling repair. We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair efficiencies ranging from high to low. We identify three dominant DNA bending states [slightly bent/unbent (U), intermediately bent (I), and significantly bent (B)] and find that the kinetics of interconverting among states varies widely for different complexes. The increased stability of MutS-mismatch/IDL complexes is associated with stabilization of U and lowering of the B to U transition barrier. Destabilization of U is always accompanied by a destabilization of B, supporting the suggestion that B is a "required" precursor to U. Comparison of MutS and MutS-E41A dynamics on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates the changes in base-base hydrogen bonding that are required to achieve the U state, which has been implicated in repair signaling. Taken together with repair propensities, our data suggest that the bending kinetics of MutS-mismatched DNA complexes may control the entry into functional pathways for downstream signaling of repair.

Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation.

Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator.

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription.

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.

Long-range architecture in a viral RNA genome.

We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10-45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5' and 3' untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simplest of the icosahedral viruses but, nonetheless, has an RNA genome with a complex higher-order structure that likely reflects high information content and an evolutionary relationship between RNA domain structure and essential replicative functions.

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D).

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.