Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

Abstract

DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.

Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE)

“…Watching DNA-binding proteins interact with DNA substrates in real-time at the single-molecule level illuminates how proteins detect and bind their targets at extraordinary detail. Key information about binding stoichiometry, order of assembly and disassembly, and how proteins diffuse to find their DNA targets are gained through single-molecule analysis (1)(2)(3). Various imaging techniques and optical platforms have been employed to resolve fluorescent proteins to the single-molecule level, but most of these techniques cluster into two broad categories -studies performed with purified proteins with defined conditions (1,4,5) or studies performed in living cells (6)(7)(8).…”

Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers

“…A central component of any single-molecule experiment designed to understand the mechanism of action of proteins that interact with DNA is the DNA construct itself (Bocanegra, Plaza, Pulido, & Ibarra, 2021;Dulin, Lipfert, Moolman, & Dekker, 2013;Kong & Greene, 2021;Ordu, Lusser, & Dekker, 2016;Spinks, Spenkelink, Dixon, & van Oijen, 2021;Yodh, Schlierf, & Ha, 2010). Moreover, the production of dedicated DNA constructs has been critical to unveil their mechanical properties and how these modulate the activity of processing enzymes; information that is hardly achievable using bulk methods (Basu, Bobrovnikov, & Ha, 2021;Brahmachari & Marko, 2018;Bustamante, Bryant, & Smith, 2003;Camunas-Soler, Ribezzi-Crivellari, & Ritort, 2016;Marin-Gonzalez, Vilhena, Perez, & Moreno-Herrero, 2021).…”

Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)

“…Watching DNA-binding proteins interact with DNA substrates in real-time at the single-molecule level illuminates how proteins detect and bind their targets at extraordinary detail. Key information about binding stoichiometry, order of assembly and disassembly, and how proteins diffuse to find their DNA targets are gained through single molecule analysis [1][2][3] . Various imaging techniques and optical platforms have been employed to resolve fluorescent proteins to the single-molecule level, but most of these techniques cluster into two broad categories -studies performed with purified proteins with defined conditions [4][5][6] or studies performed in living cells [7][8][9] .…”