Searching for DNA Damage: Insights From Single Molecule Analysis

Abstract

DNA is under constant threat of damage from a variety of chemical and physical insults, such as ultraviolet rays produced by sunlight and reactive oxygen species produced during respiration or inflammation. Because damaged DNA, if not repaired, can lead to mutations or cell death, multiple DNA repair pathways have evolved to maintain genome stability. Two repair pathways, nucleotide excision repair (NER) and base excision repair (BER), must sift through large segments of nondamaged nucleotides to detect and remove rare base modifications. Many BER and NER proteins share a common base-flipping mechanism for the detection of modified bases. However, the exact mechanisms by which these repair proteins detect their damaged substrates in the context of cellular chromatin remains unclear. The latest generation of single-molecule techniques, including the DNA tightrope assay, atomic force microscopy, and real-time imaging in cells, now allows for nearly direct visualization of the damage search and detection processes. This review describes several mechanistic commonalities for damage detection that were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA. We also discuss important findings that DNA repair proteins within and between pathways cooperate to detect damage. Finally, future technical developments and single-molecule studies are described which will contribute to the growing mechanistic understanding of DNA damage detection.

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

“… a , A structural model of PARP1 bound to nicked DNA with YFP tag (PDB codes 3ED8 and 4KLO) generated as in this reference 1 . b , A schematic of the DNA suspended between streptavidin beads containing 10 discrete nicks from the nickase Nt.BspQI.…”

“…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] .…”

Reactive Oxygen Species in the Adverse Outcome Pathway Framework: Toward Creation of Harmonized Consensus Key Events

“…ROS cause a variety of DNA lesions and the resulting cellular responses involve complex crosstalk between multiple repairs and signaling pathways ( Kay et al, 2019 ). ROS induce DNA damage such as the formation of 8-oxoguanine ( Kay et al, 2019 ; Schaich and Van Houten, 2021 ), the most abundant oxidative DNA lesion, and other types of nucleotide oxidation, deamination, and lipid peroxidation-derived adducts. DNA repair pathways for oxidative DNA lesions include base excision repair and to a lesser extent, nucleotide excision repair ( Freudenthal et al, 2017 ).…”

Searching for New Z-DNA/Z-RNA Binding Proteins Based on Structural Similarity to Experimentally Validated Zα Domain

“…Local DNA structures, also called ‘non-B’ DNA structures, have been recognised as important regulators of many fundamental regulatory processes, including replication [ 1 ], transcription [ 2 ], translation [ 3 ], epigenetics [ 4 ], DNA damage repair [ 5 , 6 , 7 ], genome evolution and rearrangement [ 8 ]. Negative supercoiling of DNA and protein binding can increase the stability of local DNA conformation and/or induce conformational changes that give rise to various alternative DNA structures, the best-described being cruciforms [ 7 ], Z-DNA/Z-RNA [ 9 , 10 ], triplexes [ 11 ] and quadruplexes [ 12 ].…”