Ultrafast structural fluctuations and rearrangements of water's hydrogen bonded network

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007."December 2006." Vita.Includes bibliographical references.Aqueous chemistry is strongly influenced by water's ability to form an extended network of hydrogen bonds. It is the fluctuations and rearrangements of this network that stabilize reaction products and drive the transport of excess protons through solution. Experimental observations of the dynamics of the hydrogen bonded network are difficult because (1) the timescales are exceedingly fast with relevant fluctuations occurring on a tens of femtosecond period and (2) the experimental probe must be sensitive to the local hydrogen bonded structure. In this thesis I address these experimental challenges through the development of ultrafast nonlinear infrared spectroscopy of the OH stretch of HOD in D20. The frequency of the OH stretch, OH, is sensitive to the configuration of the hydrogen bonded pair. Therefore, time-dependent changes in OoH can be correlated with changes in the hydrogen bonded geometry. I describe how broadband homodyne echo and polarization-dependent pump-probe experiments can be utilized to separate the contributions of spectral diffusion, vibrational relaxation and molecular reorientation. These experiments observe the underdamped motion of the hydrogen bonded pair and the librational motion of the OH dipole on the 180 and 50 fs timescales, respectively.(cont.) These dynamics occur on a relatively local (i.e. molecular) length scale. At times greater than ~300 fs the experiments observe signatures of a kinetic regime. No longer can the spectral relaxation be ascribed to a clear molecular motion. Instead, the decay originates from the collective reorganization of many molecules. Two dimensional infrared spectroscopy (2D IR) is applied to further investigate the mechanism of hydrogen bond rearrangement. 2D IR is an optical analogue of multidimensional NMR. As a correlation spectroscopy, time dependent changes in 2D IR line shapes track how vibrational oscillators relax from one frequency to another. I describe two methods of acquiring high fidelity 2D line shapes at wavelengths of 3 gtm. Both methods utilize a HeNe laser as a frequency standard and balanced detection of the signal field. Spectral diffusion is found to dominate the evolution of the 2D line shapes of the OH stretch up to the vibrational lifetime of 700 fs. At times beyond this point the line shapes change substantially, indicating population relaxation out of the v= 1 state and the formation of a spectroscopically distinct vibrationally excited ground state. Frequency dependent relaxation of the 2D IR line shapes reveals that molecules in hydrogen bonded and non-bonded configurations experience qualitatively different fluctuations.(cont.) Non-bonded configurations are found to return to band center on -100 fs timescale indicating that these configurations are inherently unstable. Hydrogen bonded oscillators undergo underdamped oscillations at the hydrogen bond stretching frequency before subsequent barrier crossing. Hydrogen bonding not only affects COOH. The transition dipole, gi, is modulated by the hydrogen bonding interaction, resulting in higher oscillator strength for strong hydrogen bonds. I describe how modeling the temperature dependent behavior of IR and Raman line shapes in combination with nonlinear IR spectroscopies can extract the frequency dependent magnitude of gi. The variation in the transition dipole with frequency is found to be roughly linear on resonance but is found to be strongly nonlinear for weak hydrogen bonds on the high frequency side of the OH line shape.by Joseph J. Loparo.Ph.D

Multistep assembly of DNA condensation clusters by SMC

SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion and DNA repair. It remains unclear how SMCs structure chromosomes and how their mechanochemical cycle regulates their interactions with DNA. Here we used single-molecule fluorescence microscopy to visualize how Bacillus subtilis SMC (BsSMC) interacts with flow-stretched DNAs. We report that BsSMC can slide on DNA, switching between static binding and diffusion. At higher concentrations, BsSMCs form clusters that condense DNA in a weakly ATP-dependent manner. ATP increases the apparent cooperativity of DNA condensation, demonstrating that BsSMC can interact cooperatively through their ATPase head domains. Consistent with these results, ATPase mutants compact DNA more slowly than wild-type BsSMC in the presence of ATP. Our results suggest that transiently static BsSMC molecules can nucleate the formation of clusters that act to locally condense the chromosome while forming long-range DNA bridges

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera

Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (~2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ~50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2)

Exchange between Escherichia coli polymerases II and III on a processivity clamp

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies