Figure 8: Proposed model of DNA lesion detection in global genomic NER. XPA (B, D): Xeroderma pigmentosum complementation group A (B, D). Image was reproduced from Kusakabe et al. (2019) ³⁰¹. License at https://creativecommons.org/licenses/by/4.0/.

Figure 9: ROS-induced DNA excision repair mechanisms. BER: base excision repair; NER: nucleotide excision repair. Image was reproduced from Lee and Kang (2019) ³¹². License at https://creativecommons.org/licenses/by/4.0/.

Base Excision Repair (BER)

The BER pathway represents a highly conserved indispensable DNA repair mechanism, exclusively practicing in resolving non-bulky single-base lesions that do not affect the DNA architecture seriously. These lesions comprise small base modifications generated by spontaneous base alkylation and deamination as well as ROS-mediated oxidation and abasic single base damage, all of which harboring a prominent pro-mutagenic and/or cytotoxic potential. These base modifications occur in accordance with the normal cell metabolism or by exposure to exogenous stressors (see above). BER can be subgrouped into two pathways, the short-patch and the long-patch repair pathway of BER ³¹³. The induction of the respective pathway not only depends on the cell cycle phase but also on the type of the abasic (AP) site primarily created and, in particular, on the origin and type of the lesion. For instance, the short-patch BER replaces single nucleotide lesions as occurring in the G1 phase, while the long-patch BER removes two to thirteen nucleotide lesions during S or G2 phase. Lesion detection and repair are mediated by specific DNA glycosylases, mono- and bifunctional, all of which harboring a common mode of action for lesion detection: i) removal of the modified base from the DNA helix, thus allowing for a precise recognition of even secondary base modifications; ii) cleavage of an N-glycosidic bond, thereby discharging a free base and generating an abasic site ³¹⁴. While monofunctional glycosylases bear a gycosylase activity only, bifunctional gycosylases are characterized by an additional intrinsic β-lyase activity ³¹⁵. Nevertheless, the processes following DNA glycosylase action are typical to the BER mechanism regardless of the nature of the glycosylase. Base elimination by a glycosylase induces the formation of an abasic site in the DNA molecule which is subsequently metabolized by an AP endocuclease. In short-patch BER, the abasic site activates the AP endonuclease 1 (APE1) which hydrolyzes the 5’-phosphodiester bond to the abasic site and creates a 3′-OH and a 5′-2-deoxyribose-5′-phosphate (5′-dRP) end. DNA polymerase β (Pol β) uses 3’-OH in order to fill this repair gap up via the process of template-directed synthesis ³¹⁶. Pol β also trims the 5’-dRP end by its intrinsic dRP-lyase activity. In long-patch BER, the repair of more than one nucleotide involves the concerted action of flap endonuclease 1 (FEN1) and DNA ligase 1 (LIG1)-mediated ligation in order to eliminate the gap (Figure 9) ^{312, 317, 318}. There are emerging data to show that poly(ADP-ribose) polymerase 1 (PARP-1), a chromatin-associated enzyme involved in maintaining genomic stability, also plays a crucial role in the repair of single-strand breaks and damaged purine bases by a sub-pathway of BER ^{314, 319}. Both sub-pathways of BER can be found in mitochondria as well, where DNA polymerase γ (Pol γ) catalyzes the filling-up of repair gaps thus highlighting the importance of this repair mechanism in the maintenance of overall genomic stability ^{320, 321}.

Mismatch Repair (MMR)

Mismatch repair (MMR) represents a conserved and eminent DNA repair pathway contributing to DNA replication fidelity, mutation prevention, and genomic stability ³²². Base mismatches in DNA are among the critically important alterations of the information content and can result from replication errors, recombination between heteroallelic parental DNAs, and nucleotide damage induced by chemical and physical stressors ³²³. In humans, recognition of DNA mismatches is facilitated by the heterodimeric MutSα complex consisting of the DNA mismatch repair proteins MSH2 (Mutator S homolog 2) and MSH6. A further complex, the heterodimer MutSβ composed of MSH2 and MSH3, exclusively detects large insertion/deletion mismatches ³²⁴. After lesion detection by the MutS complex, MutS moves along the DNA in an ATP-dependent manner enabling interaction with down-stream MMR mediators ³²⁵. Recent evidence suggests that MutS interacts with MutL at the mismatch and, thus, avoids formation of a sliding clamp ³²⁶. Afterwards, the heterodimeric MutL complexes, preferentially MutLα (MLH1/PMS2) or, alternatively, MutLβ (MLH1/MLH3), are recruited and cleave the DNA close to the mismatch via their intrinsic endonuclease activity. The resulting nick plays a crucial role in mismatch excision as it is utilized by the 5’-exonuclease 1 (Exo1) as a DNA degradation origin behind the lesion ³²⁷, thereby creating a single-stranded gap that is stabilized by replication protein A (RPA) ³²⁸. This gap is finally filled-up and ligated in conjunction with DNA polymerase δ (Pol δ), the clamp loader RFC, HMGB1 (high mobility group box 1 protein) and DNA ligase 1 (LIG1) ^{323, 328-330}. A growing body of evidence indicates that the MMR genes are down-regualted in the tumor microenvironment, under inflammatory conditions and after exposure to environmental stressors ^331-335. Of note, deficiencies in MMR impair microsatellite stability by inducing hypermutation at genomic microsatellites and thus represent a prognostic factor for survival in many cancers ^336-338.

Double-strand Break Repair (DSBR)

The most cytotoxic variant of DNA damage is represented by DNA double-strand breaks (DSB) that are crucially involved in genome destabilization and cell death ³³⁹. DSB may be formed by both, exogenous factors such as chemical and physical stressors as well as endogenously during normal cell metabolism and DNA replication ²⁹⁵.  If left unrepaired or inadequately repaired, DSB give rise to chromosomal aberrations as being a possible source for several human diseases ⁴⁷. Consequently, organisms have developed several mechanisms to resolve DSB. Amongst them, homologous recombination (HR) and non-homologous end joining (NHEJ) have been reported as being the two main pathways for DSBR. However, DSB can also be removed by additional repair mechanisms, including single-strand annealing ^{5, 293} as well as backup end joining and microhomology-directed repair, the latter occurring even in the total absence of HR and NEHJ ^{297, 298, 340}.

– Non-Homologous End Joining (NHEJ)

The non-homologous DNA end joining (NHEJ) pathway constitutes the dominant mechanism of DSBR in all mammalian cells and is putatively error-prone ²⁹⁸. Herein, the tumor suppressor p53-binding protein 1(53BP1) represents a key regulator as it serves as an interaction platform for numerous DSBR proteins (for a review see Mirza-Aghazadeh-Attari et al., 2019 ³⁴¹; Parnier and Boulton, 2014) ³⁴². In the initial phase, DSB are recognized by the heterodimeric XRCC5/6 (X-ray repair cross complementing 5/6) complex, also known as Ku70/Ku80 complex ³⁴³, which recruits further NHEJ mediators such as DNA-dependent protein kinase (DNA-PK) ³⁴⁴, XRCC4 ³⁴⁵, DNA ligase 4 (LIG4), XLF (XRCC4-like factor) ³⁴⁶, and APLF (aprataxin and polynucleotide kinase-like factor) ³⁴⁷. DNA-PK has been reported to activate XRCC4 which culminates in the stabilization and fixing of the broken DNA ends ³⁴⁸ followed by DNA end processing through the action of DNA nuclease artemis, polynucleotide 5′-kinase 3′-phosphatase (PNKP), APLF, DNA helicase WRN, XRCC5/6 (Ku70/Ku80), Flap endonuclease 1 (FEN1), and the oligomeric MRN (Mre11-Rad50-Nbs1) complex ^349-353. The process is completed by filling of the remaining gaps by family X polymerases and LIG4-mediated end joining ^{350, 354, 355}.

– Homologous Recombination (HR)

Homologous recombination (HR) embodies an error-free DNA repair tool, which predominates all through S and G2 phases of the cell cycle, making DNA more accessible for the cell and thus allowing to copy intact DNA sequences in trans in order to resolve DSB ^{356, 357}. The HR pathway comprises several sub-pathways that use DNA strand invasion and template-directed DNA repair synthesis enabling an effective lesion repair ³⁵⁸. In the initial step, the oligomeric MRN complex detects and binds to DSB followed by the recruitment of the serine/threonine kinase ATM (ataxia telangiectasia mutated) and the histone acetyltransferase Tip60 (Tat-interactive protein 60) ^{359, 360}. After Tip60-mediated acetylation and activation of ATM, the key histone variant H2AX is rapidly phosphorylated (now termed γ-H2AX) by various protein kinases such as ATM, ATR (ATM- and RAD3-related), and DNA-PK ^{361, 362}. Phosphorylated H2AX serves the function of an adapter molecule for the mediator of DNA damage checkpoint 1 (MDC1), a central player in DDR ^363-365. Phosphorylated MDC1 subsequently recruits the ubiquitin E3 ligases RNF8 and RNF168 that in turn ubiquitinate γ-H2AX ³⁶⁶. Ubiquitinated γ-H2AX acts as an adapter for several DSBR enzymes (BRAC1, 53BP1, RAD51) ^{367, 368} and chromatin-modifying complexes (SWR1, INO80) ^{369, 370}.

In the next step, the 5’-ends of DSB are excised endonucleolytically by MRN, Exo1 or BLM in order to form 3’-overhangs ³⁷¹. The addition of RAD51 generates a nucleoprotein which invades an adjacent duplex DNA and creates a D-loop conformation ^{372, 373}. The second 3’-overhang has the capacity to anneal with the shifted DNA strand at the hinge region. The 3’-OH ends utilize the intact duplex as a template for D-loop extension as part of the de novo-DNA synthesis ³⁷⁴. As a final result, two Holliday junctions (HJ) are generated that are preferentially dissolved by the BLM-TOPOIII-RMI1-RMI2 (BTR) complex. On the other hand, single HJ are cleaved by structure-selective endonucleases known as HJ resolvases ³⁷⁵.