Chunhua Xu

Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.

Pif1 is a force-regulated helicase

Pif1 is a prototypical member of the 5′ to 3′ DNA helicase family conserved from bacteria to human. It has a high binding affinity for DNA, but unwinds double-stranded DNA (dsDNA) with a low processivity. Efficient DNA unwinding has been observed only at high protein concentrations that favor dimerization of Pif1. In this research, we used single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) to study the DNA unwinding activity of Saccharomyces cerevisiae Pif1 (Pif1) under different forces exerted on the tails of a forked dsDNA. We found that Pif1 can unwind the forked DNA repetitively for many unwinding-rezipping cycles at zero force. However, Pif1 was found to have a very limited processivity in each cycle because it loosened its strong association with the tracking strand readily, which explains why Pif1 cannot be observed to unwind DNA efficiently in bulk assays at low protein concentrations. The force enhanced the unwinding rate and the total unwinding length of Pif1 significantly. With a force of 9 pN, the rate and length were enhanced by more than 3- and 20-fold, respectively. Our results imply that the DNA unwinding activity of Pif1 can be regulated by force. The relevance of this characteristic of Pif1 to its cellular functions is discussed.

Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions

Research on the dynamics of single-membrane proteins remains underdeveloped due to the lack of proper approaches that can probe in real time the proteins insertion depth in lipid bilayers. Here we report a single-molecule visualization method to track both vertical insertion and lateral diffusion of membrane proteins in supported lipid bilayers by exploiting the surface-induced fluorescence attenuation (SIFA) of fluorophores. The attenuation follows a d−4 dependency, where d is the fluorophore-to-surface distance. The method is validated by observing the antimicrobial peptide LL-37 to transfer among five transmembrane positions: the surface, the upper leaflet, the centre, the lower leaflet and the bottom of the lipid bilayer. These results demonstrate the power of SIFA to study protein-membrane interactions and provide unprecedented in-depth understanding of molecular mechanisms of the insertion and translocation of membrane proteins.

Method used to measure interaction of proteins with dual-beam optical tweezers

In the force measurement of protein-protein interaction, proteins are usually attached to microbeads, so the coated beads serve as both handles and force transducers. Due to the short interaction distance between proteins, the beads are usually close enough to each other. When dual-beam optical tweezers and quadrant photodiode detector are used to investigate the interaction of proteins, it is found that the signal of detected beads is greatly affected by adjacent beads. Analysis reveals that the contribution of two beads to the quadrant detector signal is independent. A method for extracting the real interaction signal from a disturbed one is presented. Based on this method, interaction between microtubules and AtMAP65-1 is measured. The results show that this method is useful for measuring short-distance interaction with the precision of piconewton and nanometer scales.

Asynchrony of Base-Pair Breaking and Nucleotide Releasing of Helicases in DNA Unwinding.

Helicases harness the energy of nucleotide triphosphate hydrolysis to unwind double-stranded DNA (dsDNA) in discrete steps. In spite of intensive studies, the mechanism of stepping is still poorly understood. Here, we applied single-molecule fluorescent resonant energy transfer to characterize the stepping of two nonring helicases, Escherichia coli RecQ ( E. coli RecQ) and Saccharomyces cerevisiae Pif1 (ScPif1). Our data showed that when forked dsDNA with free overhangs are used as substrates, both E. coli RecQ and ScPif1 unwind the dsDNA in nonuniform steps that distribute over broad ranges. When tension is exerted on the overhangs, the overall profile of the step-size distribution of ScPif1 is narrowed, whereas that of E. coli RecQ remains unchanged. Moreover, the measured step sizes of the both helicases concentrate on integral multiples of a half base pair. We propose a universal stepping mechanism, in which a helicase breaks one base pair at a time and sequesters the nascent nucleotides and then releases them after a random number of base-pair breaking events. The mechanism can interpret the observed unwinding patterns quantitatively and provides a general view of the helicase activity.

Bloom’s syndrome protein unfolding G-quadruplexes in two pathways*

The Bloom helicase (BLM) gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes (G4), which are non-canonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer (smFRET) to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action: when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.

Unbinding strength between C-terminal segment of AtMAP65-1 and microtubule studied with dual-optical tweezers

It has been reported that AtMAP65-1 dimer induces the formation of large microtubule bundles by forming cross-bridges between microtubules. More important, it has been reported that the C terminus of AtMAP65-1 contributes to microtubule binding and the N terminus to AtMAP65-1 dimerization. The interaction between microtubule and AtMAP65-1 is crucial since it can directly affect mechanical properties of microtubules and further the organizations and some functions of microtubules. In order to quantitatively know the interaction between microtubule and the C-terminal segment of microtubule associated protein (AtMAP65-1), we measure the unbinding force between them with dual-optical tweezers. Force histograms reveal quantized force distributions. Based on Bell-Evans-model of multiple bonds, histograms are fitted and kinetic parameters are obtained. The most probable unbinding force for the single bond is 20.0plusmn1.1 pN. Position of transition state of the bond is given to be Deltax=2.1 plusmn 0.1 nm. Intrinsic dissociation rate constant k0 is 0.0002plusmn0.00005 s-1.