Oxidative DNA Damage: Causes & Consequences of Genomic Instability - Research Paper

Introduction

According to Cheng et al. (1992), oxidative DNA damage leads to mutagenesis if left unrepaired. The oxidative DNA damage caused by the lesions produced from the attacks of ROS is required to be removed and repaired to protect cells against the consequences of genomic instability. BER pathway becomes the most important pathway which protects cells against many kinds of oxidative DNA damages (David, O'Shea, and Kundu 2007, 941; Sander and Wilson 2005, 2; Cooke, Evans, Dizdaroglu, and Lunec, 2003, 1195). Therefore, it is critical to assess how to suppress the oxidation DNA damage.

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The current body of the literature identifies OxyR genes as a promising suppressor of oxidative DNA damage. Importantly, OxyR and its regulon genes is one of the vastly studied defense mechanism against oxidative DNA damage. OxyR regulates transcription of oxidative genes either through repression or activation of genes at raised levels of H2O2. Based on these findings, it is critical to investigate the role of OxyR regulon genes in suppressing oxidative DNA damage. In doing so, microbiologist can discover new pathways to deal with illnesses associated with oxidative DNA damage. On the same note, oxidative stress is a result of overproduction in ROS due to enzymatic non-enzymatic antioxidants deficiency. Many studies have been carried out to comprehend the mechanism producing oxidative DNA damage by hydrogen peroxide (H2O2) which is converted to OH by Fenton reaction. Production of H2O2 in E. coli cells occurs mainly by the autoxidation of flavor-enzymes, whereas redox charges the stable H2O2 to the reactive OH at the expense of oxidizing ferrous iron to ferric iron. High concentration of H2O2 may cause oxidative DNA damages; however, E. coli has its own defensive mechanisms to reduce the H2O2 concentration, maintaining in vivo H2O2 concentration at a very low level. Therefore, the current study seeks to investigate the involvement of OxyR regulon genes in suppression of oxidative DNA damage via iron regulation in Escherichia coli. The findings provide crucial insights regarding oxidative DNA damage and how to suppress it. Therefore, the current study offers much-need empirical studies on how to deal with the detrimental effects of oxidative DNA damage.

Results

The results provide crucial insights regarding the involvement of OxyR regulon genes in oxidative DNA damage. The potential of OxyR as a suppressor in oxidative DNA damage is vast, as indicated in previous studies. Therefore, as part of the analysis, the oxyR deletion mutant was first constructed by P1 transduction into the defective repair system strain DmutM DmutY to prove that OxyR is a major stress regulator. The strain was then subjected to H2O2 sensitivity test by the disk diffusion assay. According to the results, the triple mutant strain DoxyR DmutM DmutY showed hypersensitivity in comparison with the DmutM DmutY strain. This result suggests that OxyR is crucial in regulating extreme stress condition, particularly in the presence of elevated H2O2 condition. Further, the deletion mutant strain was complemented with a plasmid harbouring. Consequently, the oxyR gene and its native promoter (DoxyR DmutM DmutY pBR322/oxyR), the H2O2 resistance returned almost to the level of the wild type strain (DmutM DmutY). This shows that OxyR plays an important role in the cell's defence mechanism against exogenous H2O2 treatment.

The results regarding the involvement of OxyR regulon genes in oxidative DNA also focused on observing growth phenotype for the oxyR mutant. Results showed that the mutant strain was a much slower grower both in LB and M9+glucose minimal media (Table 1). In addition, the oxidative DNA damage level was increased in the DoxyR DmutM DmutY strain in comparison with the DmutM DmutY strain by 2.7-folds and 14.2-folds in LB media and M9+glucose minimal media respectively. This finding confirms that the oxyR gene is a major key player in reducing oxidative DNA damage. Since OxyR is a transcriptional dual regulator for its regulon members, the disruption of the oxyR gene causes a detrimental effect as shown in the elongation period of growth and the increased in mutation frequency.

Table 1: Growth phenotype of mutant DoxyR grown in different growth conditions. (Growth time is measured for the strain to form colonies of 1.5 mm in size in the respective media. A slow growth phenotype is indicated by a longer time while a rapid growth is indicated by a shorter time for the strain to form the 1.5 mm colony size).

According to the findings, the mutation frequency of the oxyR mutant increased exponentially by more than 10 times in M9+glucose minimal media. At this point, it is important to note that cells use glucose as the carbon source for growth in minimal media with glucose in contrast to the utilization of amino acids in LB media. As indicated in a previous study by Gonzalez-Flecha and Demple (1995), the growth of cells in the minimal media utilized the catabolite repression pathway which yields a higher production of H2O2 by the consumption of energy in the cells and the leakage of electron in the respiration chain (Gonzalez-Flecha and Demple 1995). Therefore, the use of M9+glucose mimics an environment of high intracellular H2O2 level which also causes the increase of oxidative DNA damages. Besides that, it has been shown in previous studies of our lab members, the oxidative DNA damage increased in different folds when DmutM DmutY were grown in minimal media with different carbon sources. Hence, as shown in the mutation frequency of DoxyR DmutM DmutY, the oxidative DNA damage level increased tremendously in comparison to only the DmutM DmutY strain when the mutant strain was grown in M9+glucose minimal media.

Discussion

As stated in the literature review, OxyR gene as an oxidative DNA damage suppressor is immense. To demonstrate OxyR gene plays a role at regulating oxidative stress, a mutant DmutM DmutY containing OxyR deletion was subjected to H2O2 sensitivity test by the disk diffusion assay, however a triple mutant mutant strain DoxyR DmutM DmutY demonstrated hypersensitivity in comparison with the DmutM DmutY strain. Moreover oxyR mutant demonstrated slower growth at both in LB and M9+glucose minimal media. DNA damage increased in the DoxyR DmutM DmutY strain in comparison with the DmutM DmutY strain by 2.7-folds and 14.2-folds in LB media and M9+glucose minimal media respectively.

To determine the genes in the regulon that play a major role at suppressing oxidative DNA damage, mutant in the regulon demonstrating the highest reactivity towards oxidative stress from the H2O2 sensitivity disk diffusion, were selected. From Zheng et al (2001, 4562-4570.) microarray study, eight major genes that are highly induced by OxyR were chosen. katG deficient strain and fur deficient strain showed the highest H2O2 sensitivity. katG mutant showed H2O2 with a 38.5 mm inhibition zone, that was almost equivalent to that of the oxyR mutant with 38.7 mm inhibition zone. This result supportd other studies that KatG is the major detoxifier mediating redox agents such as H2O2 (Greenberg & Demple, 1988, 2611-2617). Fur gene deletion also showed H2O2 sensitivity of 34.9 mm inhibition zone despite this level of sensitivity, it is less than the katG deletion strain. Thus Fur gene only play a role when cell undergoes stressful conditions. Though Fur gene is not involved in scavenging for H2O2, stress induces redox reaction that sequentially activates iron regulation through Fur function.

From the above results KatG plays a crucial role in oxidative stress regulation. In determining the level of oxidative DNA damage by the mutant strain DkatG DmutM DmutY, katG deletion strain increased mutation frequency level by a significant 2.3-fold in LB media in comparison to the DmutM DmutY strain. However, in M9+glucose minimal media, the katG deletion strain did not show much effect in the level of oxidative DNA damage in comparison to the DmutM DmutY strain. KatG would be ability to suppress oxidative DNA damage by its H2O2 scavenging activity, was only seen in LB media. In M9+glucose minimal media where the H2O2 level was supposed to be higher did not affect much on the level of the oxidative DNA damage upon the katG gene deletion. This signifies that there is another important mechanism that keeps the KatG deficit in balance to control the intracellular H2O2 level or another mechanism that controls the excessive production of hydroxyl radicals that results from the intracellular H2O2.

The other alternative pathway to suppressing oxidative DNA damage may be through the hydroxyl production suppression rather than the scavenging activity of the high intracellular H2O2 level. The suppression of hydroxyl radical involves the suppression of intracellular iron levels, hence reducing the Fenton reaction from occurring. This was tested through determining the oxidative DNA damage level of the fur deletion strain which is the major iron regulator of the OxyR regulon. Fur mutant demonstrated a growth phenotype of 18 hours in LB media but was not able to grow on M9+glucose minimal media. The mutation frequency of fur deletion strain which showed increased mutation frequency of 2.5-fold in comparison with DmutM DmutY strain in LB media.It was difficult to prove that Fur which is the major iron regulator. Thus the study seeked determined the mutation frequency of the dps mutant whose main function was to sequester iron that could help reduce hydroxyl and hence suppressing oxidative DNA damage. Meanwhile, the mutation frequency results of the Ddps DmutM DmutY revealed that dps mutant increased in mutation frequency by slight significant increase of 1.6-fold on LB media while it showed a significant increase of 1.9-fold in M9+glucose minimal media in comparison to the wild type strain (DmutM DmutY). This suggests that Dps could help suppress oxidative DNA damage both in normal physiological condition and in extreme low nutrient condition.

Above results demonstrate that a single deletion strains of fur or dps only showed a minimal increase in mutation frequency. However, deletion of both fur and dps (Dfur Ddps DmutM DmutY) caused the mutation frequency to increase tremendously by 6.9-fold in comparison with the wild type strain (DmutM DmutY). The mutation frequency of Dfur Ddps DmutM DmutY also showed a significant increase by at least 2-fold in comparison with either the fur or dps deficient strain.

The YaaA protein's whose functionality is not well known except that it was proposed to facilitate in binding free unincorporated iron in the cells. Its oxidative DNA damage was investigated in the study by constructing the yaaA deletion strain in the DmutM DmutY strain. The DyaaA DmutM DmutY did not show much change in growth phenotype in comparison with the...