Abstract
Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.
Highlights
Homologous recombination (HR) is a conserved pathway for catalysing the exchange of genetic information between DNA molecules and has been shown to contribute to important cellular processes such as double-strand break repair (DSBR), rescue of collapsed or stalled replication forks, horizontal gene transfer and meiosis[1,2,3]
SM optical techniques are ideal for probing dynamic spatiotemporal processes during HR that cannot be accessed through traditional experimental approaches. We discuss their application to motor proteins, which were among the first HR proteins to be studied by SM techniques; proteins involved in assembling the presynaptic complex, namely, the ssDNAbinding proteins, which are among the first proteins to arrive at processed double-strand breaks (DSBs); and the RAD51/RecA family of DNA recombinases, which catalyse the DNA pairing and strand invasion reactions (FIG. 1)
The presynaptic complex is a crucial recombination intermediate formed when replication protein A (RPA) is replaced by RAD51 (or meiotic recombination protein DMC1/LIM15 homologue (DMC1)) in eukaryotes or when single-stranded DNA-binding protein (SSB) is replaced by RecA in bacteria (FIG. 1)
Summary
We briefly highlight some of the SM methods that have been applied to study processes related to HR; for more extensive information on SM methodologies, we refer readers to other reviews[19,20,21,22,23,24]. Applied to recombination-related reactions, epi-illumination-based techniques and TIRFM-based techniques can be used to visualize fluorescently tagged proteins as they move along nucleic acids, to monitor the colocalization of proteins on nucleic acids or to monitor the binding and dissociation kinetics of proteins. In these instances, the information acquired from the observed molecules is usually subject to standard optical resolution limits, which are typically in the order of a few hundred nanometres, resolution in the order of ~1 to tens of nanometres can be obtained by mathematical treatment of the resulting data to identify the locations of individual fluorescent spots[27]. We discuss their application to motor proteins, which were among the first HR proteins to be studied by SM techniques; proteins involved in assembling the presynaptic complex, namely, the ssDNAbinding proteins, which are among the first proteins to arrive at processed double-strand breaks (DSBs); and the RAD51/RecA family of DNA recombinases, which catalyse the DNA pairing and strand invasion reactions (FIG. 1)
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