Chemical Tools for the Study of DNA Repair

Abstract

Conspectus DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory’s work on developing chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying them. We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage. We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both in vitro and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses. To investigate cellular nucleotide sanitation pathways, we designed a series of “two-headed” nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme. We proceed to discuss the development of a “universal” probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.

Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

“…58 Recently, BER enzymes that interact with these DNA modi cations have gained signi cant attention. 59 For example, Uracil DNA glycosylase (UDG) is a BER enzyme that speci cally identi es and repairs deoxyuridine (dU), which is naturally present in genomic DNA. 60 The AP site generated by UDG from dU can be eliminated through alpha or beta elimination, 61 leading to a DNA strand break.…”

A Membrane-Confined Signal Amplification Strategy for Sensitive Monitoring of Extracellular Enzymatic Activity Upon Drug Stimulus

“…With its high programmability, easy synthesis and modification, DNA-based sensing probes have been developed for detection of a variety of biomolecules, ranging from metal ions, small molecules to biomacromolecules such as RNAs. − In particular, DNA-based fluorescent probes have been delicately designed for real-time sensing and imaging of enzymatic activities in living cells, − which have promoted the elucidation of related biochemical processes and disease diagnostics. Most recently, by engineering DNA probes with organelle-targetable ligands, in situ imaging of enzymes with subcellular specificity has been achieved. − In addition, researchers have also developed DNA-based sensor technology for specific imaging of extracellular targets such as ATP. , However, there are few DNA-based sensing probes reported for precise monitoring of extracellular enzymes, because of the following limitations: First, extracellular enzymes usually exist at a very low abundance level, thus requiring sensing probes with high sensitivity.…”

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

“…20−22 However, recent years have seen the development of more convenient, fluorescentbased methods, the vast majority of which employ nucleic acid components. 23,24 In particular, methods based on molecular beacon (MB) probes have emerged at the forefront of modern BER assays (Figure 1b). This can be attributed to their simple design and compatibility with diverse BER enzymes, especially DNA glycosylases.…”

“…The biological and clinical significance of BER has fueled the development of analytical methods for the detection of BER activity. Traditional approaches for monitoring BER activity have mainly relied on gel electrophoresis, radiation release assays, and mass spectrometry. − However, recent years have seen the development of more convenient, fluorescent-based methods, the vast majority of which employ nucleic acid components. , In particular, methods based on molecular beacon (MB) probes have emerged at the forefront of modern BER assays (Figure b). This can be attributed to their simple design and compatibility with diverse BER enzymes, especially DNA glycosylases. − Importantly, most MB probes employ a straightforward fluorescent readout, providing the ability to monitor BER activity in real time and possibly within living cells.…”