Biological Evaluation of DNA Biomarkers in a Chemically Defined and Site-Specific Manner

As described elsewhere in this Special Issue on biomarkers, much progress has been made in the detection of modified DNA within organisms at endogenous and exogenous levels of exposure to chemical species, including putative carcinogens and chemotherapeutic agents. Advances in the detection of damaged or unnatural bases have been able to provide correlations to support or refute hypotheses between the level of exposure to oxidative, alkylative, and other stresses, and the resulting DNA damage (lesion formation). However, such stresses can form a plethora of modified nucleobases, and it is therefore difficult to determine the individual contribution of a particular modification to alter a cell’s genetic fate, as measured in the form of toxicity by stalled replication past the damage, by subsequent mutation, and by lesion repair. Chemical incorporation of a modification at a specific site within a vector (site-specific mutagenesis) has been a useful tool to deconvolute what types of damage quantified in biologically relevant systems may lead to toxicity and/or mutagenicity, thereby allowing researchers to focus on the most relevant biomarkers that may impact human health. Here, we will review a sampling of the DNA modifications that have been studied by shuttle vector techniques.


Introduction
The human genome is constantly exposed to and damaged by endogenous chemicals, such as reactive oxygen species, lipid peroxidation intermediates, and alkylating agents. These electrophilic reactive chemicals, as well as environmental carcinogens and administered drugs, are known to generate various DNA adducts [1][2][3]. Some of the adducts block DNA replication or cause mutations and have been used as biomarkers to monitor the level of DNA damage or of disease progression [4][5][6]. One of the major goals for researchers is to understand the deleterious consequences of those lesions within the cell or animal. Among the different methods for studying the biological effects of the adducts, use of shuttle vectors containing a chemically defined lesion at a specific site has provided information about the biological and toxicological properties of the adduct [4,7]. The shuttle vector-based methods normally involve the steps outlined in Figure 1. Oligonucleotide synthesis: An oligonucleotide (oligo) containing a structurally defined lesion at a specific site is made either through a biomimetic route (in situ formation by direct chemical reaction, followed by HPLC purification of site-specifically modified oligo), or purely synthetically using a normal or convertible nucleoside phosphoramidite, etc. Vector construction: An ss-or ds-DNA vector containing the modified oligo is built by cutting the parent vector with one or a pair of restriction endonuclease(s), followed by ligation of the 5 -phosphorylated modified oligo. Cellular processing: The vector is transfected into different types of cells (e.g., Escherichia. coli (E. coli) or mammalian), and cellular polymerases are allowed transfected into different types of cells (e.g., Escherichia. coli (E. coli) or mammalian), and cellular polymerases are allowed to replicate or transcriptionally bypass the lesion under different repair or bypass conditions. Data analysis: DNA is extracted, amplified using PCR, and the biological outcomes are analyzed, which include the ability of the lesion/adduct to block polymerases or cause a mutation when processed by a polymerase during cellular replication. This assessment could be done by plaque or colony counting and picking with Sanger sequencing, 32 P-post labeling and thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), next-generation sequencing (NGS), etc. [4,5,[7][8][9]. The shuttle vector-based method was initially introduced by Essigmann [7,[9][10][11], further developed and utilized by Wang [4,5], Moriya [12,13], Livneh [14,15], Greenberg [16,17], Basu [18,19], Lloyd [20,21], Loechler [22,23], Fuchs [24,25], Pagès [26,27], and others. Several informative review articles have been written by these authors on designing and applying the methods. In this work, we will review a variety of DNA biomarkers or probes that have been studied using the shuttle vector techniques and briefly summarize their biological outcomes. In all cases, focus is placed on the effect of the lesion to block replication and to cause mutations. For the details regarding the formation of DNA damage and other properties of the lesions, please refer to the original literature or review articles. We apologize in advance to researchers whose work we could not include in this review. After detailed discussions on individual lesions, we will provide some perspectives on possible future directions.

Discussions on Individual Modifications
Below, we will cover modified DNA structures generated from oxidative stress, alkylation, and other processes (Figures 2-5). In the following sections, the biological effects of a certain lesion are briefly summarized. Please see Figures 2-5 for chemical structures and Table 1 for detailed information. In this work, we will review a variety of DNA biomarkers or probes that have been studied using the shuttle vector techniques and briefly summarize their biological outcomes. In all cases, focus is placed on the effect of the lesion to block replication and to cause mutations. For the details regarding the formation of DNA damage and other properties of the lesions, please refer to the original literature or review articles. We apologize in advance to researchers whose work we could not include in this review. After detailed discussions on individual lesions, we will provide some perspectives on possible future directions.

Oxidative Biomarkers
All the structures of modifications covered in this section are displayed in Figure 2 is not a strong block to replication, demonstrating greater than 80% bypass efficiency in E. coli [28]. Its mutagenic pairing with A during replication in wild type (WT)
The α-anomer of deoxynucleosides (α-dN) can be generated as a result of hydroxyl radical attack on deoxyribose [76]. All α-dNs except α-dA strongly block replication in E. coli [76]. α-dC blocks almost 99% replication and causes 72% C>A mutation [76]. α-dG also strongly blocks replication and causes 60% G>A mutation [76]. α-dT blocks almost 99% replication but it is not mutagenic in WT E. coli [76]. α-dA is not mutagenic [76]. The anticancer agent 6-thioguanine (sG) and its derivative S 6 -methylthioguanine (S 6 mG) do not block replication strongly in both E. coli and human cells [78]. sG causes 11% G>A mutation and S 6 mG causes 94% G>A mutation in E. coli [78]. sG is less mutagenic (8%) than S 6 mG (40%) in human cells as well [78]. Guanine-S 6 -sulfonic acid (SO 3 HG) is another derivative of sG [78]. It is not a strong replication block in E. coli, but it is very mutagenic, causing 77% G>A mutation [78]. The anti-HIV drug KP1212 is an analog of deoxycytidine [57]. It does not block replication in E. coli, but is mutagenic causing 10% C>T mutation [57]. Among the four 2 -deoxyxylonucleosides (xN), only xA and xG exhibit a replication block in E. coli [77]. xA is the only mutagenic lesion among the four and causes 10% A>G mutation [77]. Base J strongly blocks replication by 48%, but is not mutagenic in human cells [60].

Perspectives
In this review, we survey the biological effects of various DNA lesions or biomarkers studied by the shuttle vector techniques, allowing one to gain insight into how DNA damage or other chemically defined nucleobases are processed by polymerases and repair machinery in a natural cellular environment under physiological conditions. Among the new methods that have been developed or applied in the last decade, MS-based strategies and NGS methods have been demonstrated to be efficient for analyzing the lesion's biological outcomes. LC-MS-based methods are sensitive and accurate for quantifying the degree of lesion bypass and point mutations [4,5]. NGS techniques allow for a large-scale population analysis on many samples at the same time and provide information on a genomic perspective [4,8]. Another possible direction for using vectors as probes to analyze biomarkers is to study the mutational spectrum or mutational signature of a certain chemical or damaging agent [83][84][85][86]. LC-MS-and NGS-based analyses not only consider the biological consequences at the lesion site, but also incorporate information from the neighboring bases, such as one or two nucleotides next to the lesion site from both the 5 and 3 direction. An oligonucleotide containing the modified base can be made surrounded by nearest (and next-to nearest) randomized bases and ligated into a shuttle vector. While cellular analysis may pull out a hotspot consensus sequence for poor repair and/or mutagenic replication, this will not answer the primary question of contextual bias in adduct formation. Shuttle vector systems whereby the vector is treated with the chemical to be assessed, followed by quantification of adduct type and amount, and transfection into isogenic cells of varying repair and/or replication backgrounds may tease apart the contribution of local sequence environment to adduct formation, repair, and replication. Such vectors were used over a decade ago [87], and coupled with NGS throughput and bioinformatics, may provide enough reads to make statistically significant claims. Shuttle vectors are currently, to our knowledge, mainly DNA-based; however, one can envision use of RNA-based vectors to study the effect of modified RNA bases on cellular processes such as viral replication, translation, reverse transcription, and possibly even repair. While the role of DNA damage in toxicology focuses mainly on the direct adduction of chemical damage to DNA, pool mutagenesis has often been overlooked, and it would be interesting to leverage shuttle vector techniques to study the incorporation of modified bases from the nucleotide pool in the form of damaged DNA or from DNA-based therapeutics.