Single molecule techniques in DNA repair: A primer

Hughes, Craig D.; Simons, Michelle; Mackenzie, Cassidy E.; Houten, Bennett Van; Kad, Neil M.

doi:10.1016/j.dnarep.2014.02.003

Cited by 9 publications

(10 citation statements)

References 143 publications

(201 reference statements)

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“…Additionally, we are examining the role of UV-DDB in the recruitment of downstream NER or BER proteins at oxidative lesion sites to elucidate a role for UV-DDB in facilitating the repair process. The evidence of interplay between the two repair pathways could suggest direct interactions between BER and NER proteins, both on and off DNA, which needs further study by yeast-2-hybrid screens, co-IP and/or biochemical/biophysical methods, such as single-molecule in vitro techniques with a purified protein system or cell extracts to examine protein-protein and protein–DNA interactions at a defined lesion site ( 99 ). Moreover, these techniques can be used to examine, in real-time, how proteins act in unison to faithfully process DNA lesions.…”

Section: Discussionmentioning

confidence: 99%

The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage

Kumar

Raja

Houten

2020

Nucleic Acids Research

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The six major mammalian DNA repair pathways were discovered as independent processes, each dedicated to remove specific types of lesions, but the past two decades have brought into focus the significant interplay between these pathways. In particular, several studies have demonstrated that certain proteins of the nucleotide excision repair (NER) and base excision repair (BER) pathways work in a cooperative manner in the removal of oxidative lesions. This review focuses on recent data showing how the NER proteins, XPA, XPC, XPG, CSA, CSB and UV-DDB, work to stimulate known glycosylases involved in the removal of certain forms of base damage resulting from oxidative processes, and also discusses how some oxidative lesions are probably directly repaired through NER. Finally, since many glycosylases are inhibited from working on damage in the context of chromatin, we detail how we believe UV-DDB may be the first responder in altering the structure of damage containing-nucleosomes, allowing access to BER enzymes.

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Section: Discussionmentioning

confidence: 99%

The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage

Kumar

Raja

Houten

2020

Nucleic Acids Research

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“…UvrB also interacts with UvrC through a c-terminal coiled-coiled domain (DellaVecchia et al, 2007). We, working with Kad and coworkers, found that UvrB binds DNA tightropes in the presence of UvrC, but only if the two proteins are mixed together prior to adding to the DNA (Hughes et al, 2014). About 50% of these UvrBC complexes were motile on the DNA, displaying both random diffusion and anomalous 1D diffusion.…”

Section: Watching Uvra Uvrb and Uvrc Detect Damage In Real Timementioning

confidence: 95%

“…DNA in real-time at single-molecule scale (Kabata et al, 1993;Erie et al, 1994;Hughes et al, 2014). In order to visualize single protein molecules, the fundamental challenge is to achieve conditions where the fluorescence of one molecule can be differentiated from the background signal.…”

Section: Snapshots Of Ner and Ber Proteins Detecting Dna Damagementioning

confidence: 99%

Searching for DNA Damage: Insights From Single Molecule Analysis

Schaich

Houten

2021

Front. Mol. Biosci.

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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.

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“…The two most widely used methods to characterize the ability of a protein to processively scan DNA are singlemolecule fluorescence and ensemble biochemical processivity assays. In this review, we will highlight processivity-based assays and refer readers to excellent reviews on single-molecule fluorescence imaging techniques (9–11). However, it is worth discussing the advantages and limitations of both single-molecule and bulk biochemical assays and how the two techniques can complement each other.…”

Section: Methodsmentioning

confidence: 99%

“…Searching proteins can be directly observed scanning DNA molecules by using single-molecule fluorescence microscopy setups. Typically, proteins are visualized by attachment of quantum dots or small molecule fluorophores, through a variety of strategies (11–15). The DNA is visualized by an intercalating fluorescent molecule called YOYO-1 or in some cases not at all.…”

Section: Methodsmentioning

confidence: 99%

DNA scanning by base excision repair enzymes and implications for pathway coordination

Howard

Wilson

2018

DNA Repair

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Site-specific DNA binding proteins must search the genome to locate their target sites, and many DNA modifying enzymes have the ability to scan along DNA in search of their substrates. This process is termed processive searching, and it serves to decrease the search time by effectively increasing the DNA binding footprint of a protein. The repertoire of proteins capable of processive searching is expanding, highlighting the need to understand the governing principles behind this fundamental process. Many of the enzymes in the base excision DNA repair pathway are capable of processive searching. Here, we briefly summarize methodology for determining if a protein can scan DNA and highlight the discovery that the base excision repair DNA polymerase β performs a processive search. Elucidation of physical models for DNA searching has also provided a plausible mechanism for pathway coordination during repair. The ability of BER enzymes to transiently sample adjacent DNA sites while bound to their product confers accessibility to downstream enzymes and does not require protein-protein interactions for coordination.

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Single molecule techniques in DNA repair: A primer

Cited by 9 publications

References 143 publications

The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage

The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage

Searching for DNA Damage: Insights From Single Molecule Analysis

DNA scanning by base excision repair enzymes and implications for pathway coordination

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