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    • As discussed above both endogenous and exogenous lesions can

    2022-06-29

    As discussed above, both endogenous and exogenous lesions can lead to adverse health effects [43]. Damaged nucleobases can block DNA replication which is often lethal to cells [44]. The effectiveness of DNA restoration by DNA glycosylases should be closely monitored to gain an insight of the function of DNA. For instance, upon oxidatively induced DNA damages, DNA glycosylases are initiated to repair the ecopipam lesions that have been created [45]. One working principle to measure DNA repair is to have cells treated with a damaging agent, then the damaged DNA is incubated with DNA glycosylases to allow for the repair process, and finally measuring the amount of damages that remains [46]. The following sections describe such assays for the detection of the activity of DNA glycosylases. To date, many assays have been proposed to observe the repair mechanisms with much attention towards the glycosylase activities of uracil-DNA glycosylase (UDG), thymine DNA glycosylase (TDG), and 8-oxoguanine DNA glycosylase (OGG1). Uracil often appears in DNA via hydrolytic deamination of cytosine and misincorporation of bases during replication [47]. This frequent lesion is actively corrected by UDG, which excises the mismatched base as a free base from the U/A base pairing and any uracil base mismatches. The excision can be executed on either ss- or ds-DNA. This repair of uracil is completed with other endonuclease enzymes. Without UDG, the repair of uracil bases is unable to proceed, which may bring about diseases such as human immunodeficiency virus type 1 and bloom syndrome [48], [49]. Methylation of cytosine is a common feature in eukaryote genome. This process concerning the deamination of 5′-methylcytosine and 5′-hydroxymethylcytosine (a product from the oxidation of 5′-methylcytosine) respectively gives the final products thymine and 5′-hydroxymethyluracil, thus consequently leading to G/U and G/T mismatches. In addition, cytosine in DNA can also undergo deamination to form uracil, resulting in a G/U mismatch. The G/T mismatch is harder to detect since thymine is one of the four nucleobases of DNA. Leaving these mismatches unrepaired, the transition mutation from C to T, a point mutation, is inevitable which sometimes leads to diseases like cancer [50]. Fortunately, nature has its way to keep DNA in check via TDG mediated repair – through the BER scheme. Apart from the commonly methylated cytosine, another nucleobase, guanine can also be damaged. Oxidation of guanine by ROS leads to 8-oxoG which is a major DNA lesion. When 8-oxoG is left unrepaired, there is a chance of it mispairing with adenine during DNA replication, resulting in a transversion mutation from G to T [51]. This mutation due to 8-oxoG has ecopipam been shown to have a close correlation with human cancer [52]. The enzyme, 8-oxoguanine DNA glycosylase (OGG1) is known to excise this oxidation product of guanine [53], removing it from the DNA strand. The highest levels of OGG1 are found in thymus, testis, intestine, brain, and germinal center of B cells [54]. The detection and quantification of OGG1 is important as it is shown to be related to Parkinson's disease and cancer [55], [56].
    Conclusions and prospects However, most DNA glycosylase activity assays so far are conducted in vitro where the process is carried out in controlled environments outside living organisms. As such, these in vitro studies may fail to stimulate actual cellular conditions where DNA glycosylase activity can be affected by various physiological parameters [93], [94]. There is, however, a clear lack of impressive progress in the development of techniques to analyze DNA glycosylase in single cells in vivo or in real-time. There have been few attempts at developing methodologies for the analysis of DNA glycosylase activity in single cells with encouraging results [95], [96]. One such example can be found in a report by Hedge et al. in which they successfully localized NEIL1 DNA glycosylase in single cells [96]. To speculate further, should there eventually be highly selective glycosylase probes and fluorescence probes in particular, it would be plausible to develop glycosylase assays that are capable of tracking glycosylase in live cells in real time. Intracellular and in vivo assays, however, are typically more complex and costly and further work should be done to explore such in vivo testing. What appears to be the BER pathway mechanism for a glycosylase may be an inaccurate representation of what actually happens in cells. Since BER repair is known to utilize a group of DNA glycosylases and sometimes cofactors too to increase the affinity between the DNA glycosylases and the site of damage. Another limitation of these in vitro assays is the usage of exogenous agents to induce lesions in DNA in solution whereby its specificity is not guaranteed. Hence, there are foreseeable discrepancies between in vitro and in vivo assays despite using the same DNA glycosylases [97].