Single molecule studies of homologous recombination

Progress in Biophysics and Molecular Biology

2017

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DNA double-strand breaks (DSBs) disrupt the physical and genetic continuity of the genome. If unrepaired, DSBs can lead to cellular dysfunction and malignant transformation. Homologous recombination (HR) is a universally conserved DSB repair mechanism that employs the information in a sister chromatid to catalyze error-free DSB repair. To initiate HR, cells assemble the resectosome: a multi-protein complex composed of helicases, nucleases, and regulatory proteins. The resectosome nucleolytically degrades (resects) the free DNA ends for downstream homologous recombination. Several decades of intense research have identified the core resectosome components in eukaryotes, archaea, and bacteria. More recently, these proteins have been characterized via single-molecule approaches. Here, we focus on recent single-molecule studies that have begun to unravel how nucleases, helicases, processivity factors, and other regulatory proteins dictate the extent and efficiency of DNA resection in eukaryotic cells. We conclude with a discussion of outstanding questions that can be addressed via single-molecule approaches.

Section: Introductionmentioning

confidence: 99%

Eukaryotic resectosomes: A single-molecule perspective

Myler

Progress in Biophysics and Molecular Biology

2017

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“…These approaches are particularly useful for elucidating complex multi-step biochemical mechanisms and have proven especially amenable for studying protein-nucleic acid interactions. For example, single-molecule enzymology has shed critical insights into our understanding of DNA replication [1][2][3], transcription [4][5][6], chromatin remodeling [7], and DNA damage repair [8,9]. The development of single-molecule experiments in cell-free extracts [10][11][12] and within living cells [13,14] will continue to shed critical insights into all aspects of genome maintenance.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput single‐molecule studies of protein–DNA interactions

Robison

2014

FEBS Letters

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Fluorescence and force-based single-molecule studies of protein-nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell.

“…Direct observations of DNA replication (1–4), transcription (5–7), and repair (8–10)atthe single-molecule level are continuing to offer fresh insights into these complex, multi-step reactions. The knowledge gained from these studies typically could not be accessed using traditional biochemical or biophysical approaches.…”

Section: Introductionmentioning

confidence: 99%

Supported Lipid Bilayers and DNA Curtains for High-Throughput Single-Molecule Studies

Methods in Molecular Biology

Greene

2011

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Single-molecule studies of protein–DNA interactions continue to yield new information on numerous DNA processing pathways. For example, optical microscopy-based techniques permit the real-time observation of proteins that interact with DNA substrates, which in turn allows direct insight into reaction mechanisms. However, these experiments remain technically challenging and are limited by the paucity of stable chromophores and the difficulty of acquiring statistically significant observations. In this protocol, we describe a novel, high-throughput, nanofabricated experimental platform enabling real-time imaging of hundreds of individual protein–DNA complexes over hour timescales.