Figure 10: Generation of 8-OH-dG and 8-oxo-dG. 2′-deoxyguanosine (dG); 4,8-endoperoxide-2′-deoxyguanosine (1); 4R/4S 4-Hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine (2); 4R/4S spiroiminodihydantoin nucleoside (3); 8-Hydroperoxy-2′-deoxyguanosie (4); 8-oxo-7,8-dihydro-2′-deoxyguanosine (5); 8-Hydroxy-7,8-dihydro-2′-deoxyguanosyl radical (6); 7-Hydro-8-Hydroxy-2′-deoxyguanosine (7); 2,6-diamino-4-Hydroxy-5-formamidopyrimidine (FapyG) (8); 8-Hydroxy-2′-deoxyguanosine (9), oxazolone (10). Image was reproduced from Urbaniak et al. (2020) ³⁷⁷. License at https://creativecommons.org/licenses/by/4.0/.

5,6-Dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg)

Thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine, Tg) represents one of the most abundant oxidation products of thymidine ^{383, 384}. Tg is formed, on the one hand, by exogenous stressors such as chemical oxidants or ionizing radiation, and, on the other band, endogenously in the course of the aerobic metabolism (Figure 11). Almost 400 Tg molecules are formed per cell and day by oxidation of the double-bond in thymidine ³⁸⁵, and the existence of Tg in DNA has been utilized as a biomarker for oxidative stress ^{385, 386}. As Tg harbors two chiral C-atoms, four diastereomers exist: cis-(5R,6S), trans-(5R,6R), cis-(5S,6R), trans-(5S,6S) ³⁸⁷, amongst which cis-stereoisomers represent the predominant variants in nature ³⁸⁸. It has been shown previously that γ-irradiation produces equal amounts of the (5R, 6S) and (5S, 6R) cis-diastereomers ³⁸⁹, wherease oxidative modification leads to the preferential production of (5R, 6S) cis-diastereomers ³⁹⁰. Epimerization at C6 leads to interconversion between cis– and trans-diastereomers, even though the equilibrium favors the cis-diastereomers by the factor 3-5 ^{387, 391}. Although Tg is characterized by a poor mutagenic potential ³⁹², it has been identified as being a potent inhibitor of DNA replication ^393-395. Fixing of Tg occurs in conjunction with specific endonucleases such as prokaryotic Endo III and Endo VIII from E. coli as well as their eukaryotic counterparts ³⁹⁶. Similar to the mouse ortholog mNeil1, which is capable of excising both stereoisomers of Tg ³⁹⁷, the Endo III orthologs yNtg1 and yNtg2 from yeast have been shown to remove distinct Tg isomers ³⁹⁸. In addition, human thymine-DNA glycosylase (TDG), also termed G/T thymidine glycosylase, as well as methyl-CpG-binding protein 4 (MBD4) have been described to eliminate Tg from a G/Tg mismatch ³⁹⁹. More recent data raised by the group of Arthur P. Grollman revealed that the main DNA glycosylases in bacteria, yeast and mammalian cells stereoselectively remove the (5S, 6R) and (5R, 6S) isomers of Tg from duplex DNA, thereby implying the occurrence of these enzymes as being complementary pairs in their respective organisms ⁴⁰⁰. Several studies revealed that Tg is primarily removed by BER, although NER may also be involved in the repair of this respective DNA lesion ^401-403. Since G/T pairs in DNA have been reported as being efficiently repaired by the MMR system ⁴⁰⁴, it has been proposed that the MMR system might also play a crucial role in the replacement of Tg-bearing DNA ^{405, 406}. However, data raised in E. coli by Perevozchikova and collaborators indicate that Tg-DNA obviously does not function as a substrate for the DNA MMR system ⁴⁰⁷.

Pyrimidine Dimers

The UV-induced formation of pyrimidine dimers in DNA is a main detrimental mechanism in both, eukaryotic and prokaryotic cells. UV irradiation is categorized into three classes on the basis of the range of wavelength: UV-A (320–400nm), UV-B (290–320 nm), and UV-C (190–290 nm). Previous studies reported on the prominent DNA-damaging potential of UV-C as being mediated by covalently linking two adjacent pyrimidines that belong to the same DNA strand or to different DNA strands. This dimerization process predominantly generates cyclobutane pyrimidine dimers (CPD) and pyrimidine (6 – 4) pyrimidone photoproducts [(6 – 4)PP; Figure 1] ^2-8. In CPD, the two adjacent pyrimidines are covalently connected by a cyclobutane ring, while in (6 – 4)PP the C6 position of one pyrimidine is covalently attached to the C4 position of the neighboring pyrimidine. These bulky dimers lead to conformational rearrangements of the helix, thereby accounting for cell death and mutagenicity. UV-A and UV-B are also capable of inducing the formation of pyrimidine dimers, but to a much lesser extend ^{1, 4}. Numerous investigations reveal that pyrimidine dimers induce various mutations such as CG→TA, TA→CG and typical tandem CC→TT transitions ^408-410. An intriguing feature of (6 – 4)PP is represented by the fact that it can be converted by UV-B to a Dewar photoproduct, a valence isomer of (6 – 4)PP, which in turn has the capacity to regenerate the regular (6 – 4)PP isomer upon exposure to UV-C ^{6, 7}. (6-4)PP and its Dewar photo-isomer are important cytotoxic determinants of UV light contributing to sunlight carcinogenesis ⁷. Unrepaired UV lesions, however, hinder DNA replication and block normal progression of replication forks.

Figure 1. UV-induced pyrimidine dimers in DNA. (Left) Cyclobutane pyrimidine (thymine-thymine) dimer; (Right) pyrimidine (6 – 4) pyrimidone photoproduct. Image was reproduced, with permission, from Nikitaki et al. (2015) ⁶⁴⁴. License at https://creativecommons.org/licenses/by/4.0/

Organisms in all kingdoms of life have developed multiple repair pathways aiming to resolve and circumvent UV-induced DNA lesions ⁴¹¹. The plainest pathway, also known as direct reversal, utilizes photolyases that disrupt the covalent bonds between the two pyrimidines by using visible blue-light in sunlight ³. CPD photolyases are divided into class I, II, III ^{412, 413} and ssDNA photolyases ⁴¹⁴ that bear a flavin adenine dinucleotide (FAD) molecule as the key coenzyme ^{3, 415}. As outlined by Zhang et al. (2017) ⁴¹⁶, the mechanism of CPD repair can be initiated either by direct light absorption of reduced FADH⁻ or via energy transmission from the excited antenna chromophore. Afterwards, the excited FADH^−* assigns an electron to the CPD substrate which spontaneously disbands into its individual monomers, two thymine bases. In the final step, the electron turns back to the coenzyme and thus restores the enzyme activity. An alternative mechanism can be found in prokaryotes, in which a DNA glycosylase (T4 endonuclease V) specifically dissects the 5′-connected CPD base and the DNA backbone via the process of β-elimination ⁴¹⁷. In bacteria and fungi, UV damage endonuclease (Uve1p, UVED) has also been reported to cleave the 5’-connection ⁴¹⁸. The CPD process is completed by BER thus culminating in the restoration of the DNA ^{419, 420}.

The central repair pathway for UV lesions, nonetheless, is represented by the NER system. NER is found in all organisms and can be subdivided into two signaling pathways: i) global genomic NER (GG-NER), and ii) transcription-coupled NER (TC-NER). Both pathways utilize a large number of signaling proteins and differ remarkably in the first fundamental step, the detection of DNA lesions (reviewed by Kusakabe et al. (2019) ³⁰¹. NER starts with the recognition of UV lesions followed by segregation and incision of the DNA strand on both sides of the lesion, proceeding to lesion repair and subsequent de novo-DNA synthesis and ligation (summarized in ^{411, 421}). During DNA replication, bypass of UV lesions can be achieved by either homologous recombination (HR) ⁴²² or translesion synthesis (TLS) triggered by specialized DNA polymerases ^{411, 423}. TLS aims at exploiting benefits from transcription blockade mediated by the presence of DNA lesions and recognizes lesions on the transcribed strand. Various ATPases play crucial roles in the NER mechanism contributing to lesion recognition. In bacteria, lesion detection is facilitated by the ATPases UvrA and UvrB during GG-NER ⁴²¹, while in eukaryotes this process is performed by damaged DNA-binding protein 2 (DDB2), also termed Xeroderma pigmentosum complementation group E (XPE), as part of the heterodimeric UV-DDB protein in conjunction with the XPC complex ^424-426. ATP-dependent proofreading and lesion-mediated strand segregation in eukaryotes are obviously performed by XPB and XPD ATPases/helicases in the oligomeric transcription factor complex TFIIH ^303-305. After successful verification, specific nucleases are recruited for DNA incision and lesion repair. Additional information is given in section Nucleotide Excision Repair (NER).

γ-H2AX (H2AX)

H2AX is a mammalian histone variant and a member of the H2A family. As the main protein constituent of chromatin, histones are subjected to various types of post-translational modifications, amongst which phosphorylation, ubiquitination, and sumoylation play critical roles in DNA double-strand break repair (DSBR). Once DSB are formed in DNA, H2AX becomes rapidly phosphorylated at the C-terminal S139. This phosphorylation site is unique amongst the H2A histone family members and designated as “γ-phosphorylation”. Accordingly, the label “γ-H2AX” illustrates the specific phosphorylation at S139 of H2AX. γ-H2AX represents a sensitive marker for DNA DSB. As summarized by Pinder and colleagues ²⁹² (Figure 12), the induction of DSB in DNA leads to the recruitment of the MRN (Mre11-Rad50-Nbs1) complex to the damage site ^{363, 364}. It has been shown previously that constitutive ubiquitination of Nbs1 by the E3 ubiquitine ligase RNF8 (E3 ring finger protein 8)-UbcH5 (ubiquitin-conjugating enzyme H5c) during early DSBR is a prerequisite for the targeted recruitment of the MRN complex to DSB ⁴²⁷. Moreover, BMI1-RNF2 is recruited to DSB after chromobox homolog 4 (CBX4)-mediated sumoylation of BMI1 at K88 ⁴²⁸. The E3 ubiquitin ligase RNF2 (RING1b/RING2)-UbcH5 and its adapter molecule BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) catalyze the mono-ubiquitination of H2AX at K118/119 as a prerequisite for the successive targeting of the ataxia telangiectasia mutant (ATM) protein kinase and valuable formation of γ-H2AX ^{429, 430}. ATM is activated and recruited to DNA damage sites in complex with the histone acetyltransferase Tip60 ^{359, 360}. In the next step, H2AX is rapidly phosphorylated by ATM and various other protein kinases such as ATR (ATM- and RAD3-related), and DNA-dependent protein kinase (DNA-PK) ^{361, 362}.