Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System.

Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the versatility of optical trapping. Quadruple (Q)-trap optical tweezers are an excellent example of such an advance, by providing three-dimensional control over two constructs and thereby enabling for example DNA-DNA braiding. However, the implementation of fluorescence detection in such a Q-trapping system poses several challenges: (1) since typical samples span a distance in the order of tens of micrometers, it requires imaging of a large field of view, (2) in order to capture fast molecular dynamics, fast imaging with single-molecule sensitivity is desired, (3) in order to study three-dimensional objects, it could be needed to detect emission light at different axial heights while keeping the objective lens and thus the optically trapped microspheres in a fixed position.

Duplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex.

Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. TRR plays an essential role in resolving DNA replication and recombination intermediates, often alongside the helicase BLM. While the TRR catalytic cycle is known to involve a protein-mediated single-stranded (ss)DNA gate, the detailed mechanism is not fully understood.

Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches.

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair.

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping.

Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive.

Reconstitution of anaphase DNA bridge recognition and disjunction.

Faithful chromosome segregation requires that the sister chromatids be disjoined completely. Defective disjunction can lead to the persistence of histone-free threads of DNA known as ultra-fine bridges (UFBs) that connect the separating sister DNA molecules during anaphase. UFBs arise at specific genomic loci and can only be visualized by detection of associated proteins such as PICH, BLM, topoisomerase IIIα, and RPA.