Biomedical subjects

Taekjip Ha

Publications and source records attributed to Taekjip Ha.

At least 19 recordsLinked to original sources

PubMed2026

Direct visualization of MCM helicase activation and replisome coupling in situ.

Deciphering the spatial organization of molecular machines that copy the genome remains a fundamental challenge in biology. Essential for eukaryotic DNA replication, Mini-Chromosome Maintenance (MCM2-7) helicases are loaded during G1 as double hexamers (DHs) to license replication origins. Upon activation in S phase, each DH is thought to split into two single hexamers (SHs) that form the active CMG helicases and travel bidirectionally. However, the field has long been divided: biochemical and structural studies define CMG helicases as autonomous, independent motors, while genomic and cellular imaging assays suggest sister replisomes remain physically coupled within replication factories. Here, we use MINFLUX nanoscopy to localize individual MCM complexes down to nanometer precision in situ, directly resolving DHs in human cells and capturing their separation into SHs upon origin firing. We find that the resulting sister replisomes do not diffuse apart: they remain coupled at a characteristic distance of ~40 nm throughout S phase. Depletion experiments identify two distinct contributions to this coupling: local, protein-mediated tethering by the AND1 scaffold, and higher-order spatial confinement dependent on cohesin, which is dispensable for MCM loading in G1 but required to maintain coupling in S phase. By linking the nanometer-scale architecture of the replisome to the genome-wide topology of replication fountains, these findings provide direct spatial evidence that sister forks are coupled during DNA synthesis and define the molecular forces that organize replisomes within their native nuclear context.

DNA replication↗

PubMed2026

Folding a broken genome: the versatile roles of cohesin in genome maintenance.

Cohesin is a protein complex that shapes 3D genome organization through two distinct mechanisms. First, cohesin tethers replicated chromatids from DNA replication until mitosis. This process, known as sister chromatid cohesion, ensures accurate chromosome segregation and enables high-fidelity DNA repair through homologous recombination between the sister chromatids. Second, cohesin organizes the genome during interphase by dynamically extruding chromatin loops, structures that have key roles in gene regulation. Recent work has shown that, in addition to the well-established repair functions of sister chromatid cohesion, cohesin-mediated chromatin looping is closely linked to the repair of DNA double-strand breaks - one of the most toxic DNA lesions. In this Review, we discuss the central roles of cohesin in maintaining genome stability, with emphasis on the cellular response to DNA double-strand breaks. We review how dynamic loop structures facilitate signalling of repair events and promote long-range chromatin motions that underpin the repair process. Overall, its dual mode of action - cohesion and loop extrusion - positions cohesin as a central regulator of chromatin architecture and genome maintenance.

Cohesins↗

PubMed2025

Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.

A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.

DNA Breaks, Double-Stranded↗

PubMed2022

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Humans↗

PubMed2006

Nonblinking and long-lasting single-molecule fluorescence imaging.

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Carbocyanines↗

PubMed2006

Unraveling helicase mechanisms one molecule at a time.

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo.

Base Pairing↗

PubMed2006

Real-time observation of RecA filament dynamics with single monomer resolution.

RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.

Binding Sites↗

PubMed2006

Structure-based model of the stepping motor of PcrA helicase.

DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move along single-stranded DNA (ssDNA) and separate duplex DNA into its component strands, utilizing the free energy from ATP hydrolysis. The PcrA helicase from Bacillus stearothermophilus translocates as a monomer progressively from the 3' end to the 5' end of ssDNA and is one of the smallest motor proteins structurally known in full atomic detail. Using high-resolution crystal structures of the PcrA-DNA complex, we performed nanosecond molecular dynamics simulations and derived potential energy profiles governing individual domain movement of the PcrA helicase along ssDNA. Based on these profiles, the millisecond translocation of the helicase along ssDNA was described through Langevin dynamics. The calculations support a domain stepping mechanism of PcrA helicase, in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of domains 2A and 1A are synchronized with alternating mobilities of the individual domains in such a fashion that PcrA moves unidirectionally along ssDNA. By combining short timescale (nanoseconds) molecular dynamics and long timescale (milliseconds) stochastic-dynamics descriptions, our study suggests a structure-based mechanism of the ATP-powered unidirectional movement of PcrA helicase.

Adenosine Triphosphate↗

PubMed2006

Analysis of single-molecule FRET trajectories using hidden Markov modeling.

The analysis of single-molecule fluorescence resonance energy transfer (FRET) trajectories has become one of significant biophysical interest. In deducing the transition rates between various states of a system for time-binned data, researchers have relied on simple, but often arbitrary methods of extracting rates from FRET trajectories. Although these methods have proven satisfactory in cases of well-separated, low-noise, two- or three-state systems, they become less reliable when applied to a system of greater complexity. We have developed an analysis scheme that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the most likely time sequence of underlying states for each trajectory. Together with a transition density plot and Bayesian information criterion we can also determine the number of different states present in a system in addition to the state-to-state transition probabilities. Here we present the algorithm and test its limitations with various simulated data and previously reported Holliday junction data. The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct.

Algorithms↗

PubMed2006

Defocused orientation and position imaging (DOPI) of myosin V.

The centroid of a fluorophore can be determined within approximately 1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (beta) averaged 128 degrees while the LCD was in the leading state and 57 degrees in the trailing state. The angular difference of 71 degrees represents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When beta changes, the probe rotates +/-27 degrees azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.

Animals↗

PubMed2006

Bridging conformational dynamics and function using single-molecule spectroscopy.

In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase.

Adenosine Triphosphate↗

PubMed2006

Single molecule nanometronome.

We constructed a DNA-based nanomechanical device called the nanometronome. Our device is made by introducing complementary single-stranded overhangs at the two arms of the DNA four-way junction. The ticking rates of this stochastic metronome depend on ion concentrations and can be changed by a set of DNA-based switches to deactivate/reactivate the sticky end. Since the device displays clearly distinguishable responses even with a single base pair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA.

DNA↗

PubMed2006

Methanosarcina acetivorans flap endonuclease 1 activity is inhibited by a cognate single-stranded-DNA-binding protein.

The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms, Methanosarcina acetivorans harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5' region cleaved during Okazaki fragment processing. We report here that M. acetivorans RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by M. acetivorans flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.

Amino Acid Sequence↗

PubMed2005

Extreme conformational diversity in human telomeric DNA.

DNA with tandem repeats of guanines folds into G-quadruplexes made of a stack of G-quartets. In vitro, G-quadruplex formation inhibits telomere extension, and POT1 binding to the single-stranded telomeric DNA enhances telomerase activity by disrupting the G-quadruplex structure, highlighting the potential importance of the G-quadruplex structure in regulating telomere length in vivo. We have used single-molecule spectroscopy to probe the dynamics of human telomeric DNA. Three conformations were observed in potassium solution, one unfolded and two folded, and each conformation could be further divided into two species, long-lived and short-lived, based on lifetimes of minutes vs. seconds. Vesicle encapsulation studies suggest that the total of six states detected here is intrinsic to the DNA. Folding was severely hindered by replacing a single guanine, showing only the shortlived species. The long-lived folded states are dominant in physiologically relevant conditions and probably correspond to the parallel and antiparallel G-quadruplexes seen in high-resolution structural studies. Although rare under these conditions, the short-lived species determine the overall dynamics because they bridge the different long-lived species. We propose that these previously unobserved transient states represent the early and late intermediates toward the formation of stable G-quadruplexes. The major compaction occurs between the early and late intermediates, and it is possible that local rearrangements are sufficient in locking the late intermediates into the stably folded forms. The extremely diverse conformations of the human telomeric DNA may have mechanistic implications for the proteins and drugs that recognize G-rich sequences.

DNA↗

PubMed2005

Repetitive shuttling of a motor protein on DNA.

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.

Adenosine Triphosphate↗

PubMed2005

Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model.

Myosin VI walks in a hand-over-hand fashion with an average step size of 30 nm, which is much larger than its 10 nm lever arm. Recent experiments suggest that the myosin VI structure has an unfolded and flexible region in the proximal tail which makes such a large step possible. In addition, cryoelectron microscopy images of actomyosin VI show the two heads bound to the actin monomers with a broad distribution of distances, including some as close as a few nanometers. This observation, when combined with the existence of a flexible region in the structure, which takes part in stepping, challenged the hand-over-hand model. In the hand-over-hand model, the lever arm is considered to be rigid and the interhead separation should not be very different from 30 nm. We considered an alternative model in which myosin VI heads sequentially take 60 nm steps whereas the interhead separation alternates between a large and small value (x and 60 - x, where x < 30). To clarify these issues, we used a new technique, SHRImP, to measure the interhead distance of nearly rigor myosin VI molecules. Our data show a single peak at 29.3 +/- 0.7 nm, in agreement with the straightforward hand-over-hand model.

Actins↗

PubMed2005

Observing spontaneous branch migration of Holliday junctions one step at a time.

Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.

Base Pairing↗

PubMed2005

The euryarchaeota, nature's medium for engineering of single-stranded DNA-binding proteins.

The architecture of single-stranded DNA-binding proteins, which play key roles in DNA metabolism, is based on different combinations of the oligonucleotide/oligosaccharide binding (OB) fold. Whereas the polypeptide serving this function in bacteria contains one OB fold, the eukaryotic functional homolog comprises a complex of three proteins, each harboring at least one OB fold. Here we show that unlike these groups of organisms, the Euryarchaeota has exploited the potential in the OB fold to re-invent single-stranded DNA-binding proteins many times. However, the most common form is a protein with two OB folds and one zinc finger domain. We created several deletion mutants of this protein based on its conserved motifs, and from these structures functional chimeras were synthesized, supporting the hypothesis that gene duplication and recombination could lead to novel functional forms of single-stranded DNA-binding proteins. Biophysical studies showed that the orthologs of the two OB fold/one zinc finger replication protein A in Methanosarcina acetivorans and Methanopyrus kandleri exhibit two binding modes, wrapping and stretching of DNA. However, the ortholog in Ferroplasma acidarmanus possessed only the stretching mode. Most interestingly, a second single-stranded DNA-binding protein, FacRPA2, in this archaeon exhibited the wrapping mode. Domain analysis of this protein, which contains a single OB fold, showed that its architecture is similar to the functional homologs thought to be unique to the Crenarchaeotes. Most unexpectedly, genes coding for similar proteins were found in the genomes of eukaryotes, including humans. Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence.

Amino Acid Motifs↗