Many enzymes that react with specific sites in DNA exhibit the property of facilitated diffusion, where the DNA chain is used as a conduit to accelerate site location. Despite the importance of such mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method where a small molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in DNA. The “molecular clock” provides unprecedented information: the mean length for DNA sliding, the 1D sliding constant, the maximum hopping radius and time frame for DNA hopping events. In addition, the data establish that the DNA phosphate backbone is a sufficient requirement for DNA sliding.

RNA G-quadruplexes located within the 5'-UTR of mRNA are almost always known to be associated with repression of cap-dependent translation. However, in this report we present functional as well as structural evidence that sequence redundancy in a G-rich segment within the 5'-UTR of human VEGF mRNA supports a 'switchable' RNA G-quadruplex structure that is essential for IRES-mediated translation initiation. Additionally, utilization of a specific combination of G-tracts within this segment allows for the conformational switch that implies a tunable regulatory role of the quadruplex structure in translation initiation. A sequence engineered from a functionally handicapped mutant moderately rescued the activity, further indicating the importance of G-quadruplex structure for VEGF IRES-A function. This to our knowledge is the first report of a conformationally flexible RNA G-quadruplex which is essential for IRES-mediated translation initiation.

The insulin linked polymorphism region (ILPR) is known to regulate transcription of the gene coding for insulin. The ILPR has guanine rich segments, suggesting that G quadruplexes may be responsible for this regulatory role. Using mechanical unfolding in a laser tweezers instrument and circular dichroism (CD) spectroscopy, we provide compelling evidence that highly stable parallel and antiparallel G quadruplex structures coexist in the predominant ILPR sequence of (ACAGGGGTGTGGGG)(2) at a physiologically relevant concentration of 100 mM KCl. Experiments at the single molecular level have shown that unfolding forces for parallel and antiparallel structures (F(unfold): 22.6 vs 36.9 pN, respectively) are higher than the stall forces of enzymes having helicase activities. From a mechanical perspective alone, these data support the hypothesis that G quadruplexes may cause replication slippage by blocking replication process. Using the unique combination of the rupture force and the contour length measured by laser tweezers, the simultaneous determination of probable parallel and antiparallel G quadruplex structures in a solution mixture has been achieved. Jarzynski's equality analysis has revealed that the antiparallel G quadruplex is thermodynamically more stable than the parallel conformer (DeltaG (unfold): 23 vs 14 kcal/mol, respectively). On the other hand, kinetic measurements have indicated that both parallel and antiparallel structures fold rather rapidly (k(fold): 0.4 vs 0.3 s(-1), respectively), suggesting that they may be kinetically accessible for gene control. This work provides an unprecedented mechanical perspective on G quadruplex stability, presenting a unique opportunity to predict the functional consequence when motor enzymes encounter such structures.

Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double stranded DNA. Whereas single molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C) rich region complementary to G quadruplex forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5′-(TGTCCCCACACCCC)2, a predominate variant in the insulin linked polymorphic region (ILPR). We have observed two species with the change in contour length (ΔL) of 10.4 (±0.1) and 5.1 (±0.5) nm, respectively. Since ΔL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br2 footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23 % to 1.3 %) with pH 5.5 7.0, which is a well established behavior for i-motifs. In contrast to the i-motif, the formation of the second species with ΔL of 5.1 nm remains unchanged (6.1±1.6 %) in the same pH range, implying that pH sensitive C:CH+ pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the ΔGunfold of i-motif (16.0 ±0.8 kcal/mol), the decreased free energy in the partially folded structure (ΔGunfold 10.4 ± 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH+ pairs. Both ΔL and ΔGunfold argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.

Intracellular space is at a premium due to the high concentrations of biomolecules and is expected to have a fundamental effect on how large macromolecules move in the cell. Here, we report that crowded solutions promote intramolecular DNA translocation by two human DNA repair glycosylases. The crowding effect increases both the efficiency and average distance of DNA chain translocation by hindering escape of the enzymes to bulk solution. The increased contact time with the DNA chain provides for redundant damage patrolling within individual DNA chains at the expense of slowing the overall rate of damaged base removal from a population of molecules. The significant biological implication is that a crowded cellular environment could influence the mechanism of damage recognition as much as any property of the enzyme or DNA.

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine (oG) DNA glycosylase 1 (hOGG1), which repairs mutagenic oG lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a covalent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic parameters describing the DNA search of hOGG1 were derived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dynamic multidimensional diffusion paths to scan DNA.

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPRn=4). Mobilities of ILPRn=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPRn=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPRn=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPRn=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPRn=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

Nuclear human uracil–DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N–terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post–translational modifications. Although the N–terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N–terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy.