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A change of view: homologous recombination at single-molecule resolution

Key Points

  • Homologous recombination (HR) is an important pathway that enables the exchange of genetic information between DNA molecules.

  • Single-molecule (SM) optical approaches are ideal for probing dynamic spatiotemporal processes that cannot be easily studied through traditional experimental approaches.

  • SM optical techniques have uncovered many features of HR and are providing new insights into molecular mechanisms with unprecedented detail.

  • Super-resolution optical microscopy offers the ability to image fluorescent molecules inside fixed or living cells with greater spatial resolution than is possible with conventional microscopy. Its application to visualize recombination is an emerging area of tremendous potential.

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.

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Figure 1: Overview of homologous recombination.
Figure 2: Examples of single-molecule imaging methods.
Figure 3: Examples of single-molecule experiments.
Figure 4: Super-resolution imaging methods.
Figure 5: Super-resolution images of the synaptonemal complex.
Figure 6: SIM imaging of recombination structures in living cells.

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Acknowledgements

The authors thank members of the Greene laboratory for their insights during the preparation of this manuscript. This research was funded by US National Institutes of Health grant R35GM118026 and by US National Science Foundation grant MCB−1154511 (E.C.G.). L.D.T. was supported by a Pew Latin American Fellowship, the Williams Foundation and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. The authors apologize to colleagues whose works were unable to be cited owing to length limitations.

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K.K. and L.D.T researched data for the article and wrote the initial draft of the manuscript. E.C.G. reviewed and edited the manuscript before submission and prepared the final draft.

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Correspondence to Eric C. Greene.

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PowerPoint slides

Glossary

Double-strand break repair

(DSBR). An umbrella term that encompasses numerous potential pathways for the repair of a double-strand break. These pathways include non-homologous end joining, microhomology-mediated end joining, synthesis-dependent strand annealing and homologous recombination.

Horizontal gene transfer

The transfer of genetic material between the genomes of two organisms that does not occur through parent–progeny transmission. Also referred to as lateral gene transfer.

Loss of heterozygosity

A loss of one of the alleles at a given locus as a result of a genomic change, such as mitotic deletion, gene conversion or chromosome mis-segregation.

Holliday model

An early model of double-strand break repair that proposed a cross-stranded structure was the result of two single-stranded DNA breaks and accounted for both gene conversion and crossing over. While the model was later updated by Meselson and Radding, the crossover structure called the Holliday junction remains a cornerstone of recombination.

Meselson–Radding model

An update to the Holliday model that accounted for aberrant segregation in yeast tetrad analysis whereby recombination is initiated through a single double-stranded DNA break rather than two single-stranded DNA breaks. The model still employs the crossover structure first proposed by Holliday.

Synthesis-dependent strand annealing

(SDSA). A mode of double-strand break repair that proceeds through the early steps of homologous recombination but does not include second-end capture or the Holliday junction. Instead, after DNA synthesis primed by the initial captured end, the heteroduplex joint is dissociated to re-anneal with the second end of the double-strand break and serve as the template for further gap repair and resolution.

Meiotic recombination

A specialized type of homologous recombination that takes place during meiosis and is required to generate a physical linkage called a chiasma between two (non-sister) homologous chromosomes. Most, but not all, eukaryotes require the meiosis-specific recombinase meiotic recombination protein DMC1/LIM15 homologue (DMC1) to promote meiotic recombination.

Single-molecule optical microscopy

A class of techniques that use optical microscopy to study the biochemical and biophysical properties of biological molecules.

Super-resolution optical microscopy

A class of microscopy techniques used to enhance the spatial resolution of an optical microscope.

RAD51/RecA family of DNA recombinases

A highly conserved family of ATP-dependent DNA-binding proteins that promote critical steps in homologous recombination. Examples of key members of this family include bacteriophage T4 recombination and repair protein (UvsX), bacterial protein RecA, archaeal RadA, and the eukaryotic recombinases RAD51 and meiotic recombination protein DMC1/LIM15 homologue (DMC1).

Evanescent field

An oscillating electromagnetic field that is spatially concentrated at the interface between two materials with different refractive indices, for instance, glass and water. Also known as an evanescent wave. In the case of total internal reflection microscopy, the evanescent field is confined near the interface between the aqueous buffer and the glass microscope slide (or coverslip).

Single-molecule FRET

(smFRET). A technique that allows the direct measurement of distances between macromolecules of interest within a range of ~10–50 Å. Also referred to as single-pair FRET (spFRET). smFRET is commonly used with total internal reflection microscopy but can be adapted for use in many types of single-molecule imaging systems.

Presynaptic complex

A nucleoprotein complex that comprises the presynaptic single-stranded DNA and the associated protein cofactors necessary for promoting homologous recombination (HR). Key among these factors are the RAD51/RecA family of DNA recombinases, which act together with a number of associated factors to promote HR.

Crossover hotspot instigator sites

(chi sites). Specific cis-acting sites consisting of an eight-nucleotide DNA sequence (5′-GCTGGTGG-3′) that is over-represented in the Escherichia coli genome and helps regulate the properties of the RecBCD complex by controlling the activities of the RecBCD enzyme subunits RecB and RecD ATP-dependent motor proteins.

Translocation

General term used to indicate the ATP-dependent movement of a motor protein (such as a helicase or a polymerase) along a nucleic acid.

Processivity

The tendency of a helicase (or any other nucleic acid motor protein) to continue to move along a nucleic acid molecule rather than dissociating into free solution.

Nucleoprotein filaments

The helical filament structures formed by members of the RAD51/RecA family of recombinases as they bind to single-stranded DNA. They contain one protein monomer for every three DNA bases and six protein monomers per helical turn. These nucleoprotein filaments are a key component of the presynaptic complex.

Homology search

The process during which the presynaptic complex searches the genome for a double-stranded DNA sequence that is homologous to the single-stranded DNA sequence present at the processed ends of a double-strand break.

Strand invasion

A reaction catalysed by the RAD51/RecA family of recombinases, which allows for Watson–Crick pairing interactions between a single-stranded DNA molecule and the complementary strand within a homologous double-stranded DNA, resulting in displacement of the non-complementary strand.

D-Loop

The paired intermediate generated by a successful strand invasion reaction wherein the 3′ end of the invading single-stranded DNA strand is now available as a primer for DNA synthesis.

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Kaniecki, K., De Tullio, L. & Greene, E. A change of view: homologous recombination at single-molecule resolution. Nat Rev Genet 19, 191–207 (2018). https://doi.org/10.1038/nrg.2017.92

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