DNA Damage: The DNA Damage Repair (DDR) System
The human genome is permanently attacked by genotoxic and cytotoxic insults from both, endogenous and exogenous resources leading to aberrant DNA structures, thereby impacting genomic stability and, consequently, health and disease states. In order to assure regular cell metabolism, a complex network of biological processes evolved over time that prevents genomic destabilization by accumulating DNA lesions. This signaling network, termed the DNA damage repair (DDR) system and also known as DNA repair response, combines various DNA repair and DNA damage tolerance pathways as well as cell cycle checkpoint activation and transcription regulation (for a review see 5, 292-295). The DDR system comprises at least five major pathways such as homologous recombination (HR), mismatch repair (MR), non-homologous end joining (NHEJ), base excision repair (BER) and, importantly, nucleotide excision repair (NER) 293, 296. DNA single-strand breaks (SSB) are repaired by BER or NER, whereas DNA double-strand breaks (DSB) are repaired by HR or NHEJ. Alternatively, DSB can also be removed by backup end joining or microhomology-directed repair in the absence of HR and NEHJ 297, 298.
Nucleotide Excision Repair (NER)
Nucleotide excision repair (NER) is a highly complex DNA repair mechanism associated with the elimination of numerous bulky, helix-distorting DNA lesions 299, 300. 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. In GG-NER, the heterotrimeric lesion recognition factor XPC/RAD23/CETN2 detects and binds to DNA lesions characterized by a disrupted helix conformation 302. Afterwards, the oligomeric transcription factor TFIIH is recruited and verifies the presence of a critical lesion in conjunction with the ATPase/helicase subunits XPB and XPD 303-305. Herein, XPD translocates along the opposite DNA strand until a critical lesion is reached which blocks its translocation, thus enabling the intermediate repair complex to pass to dual incisions (Figure 8) 301. The DNA repair protein XPA (Xeroderma pigmentosum complementation group A) functions as a crucial factor in DNA damage recognition as it harbors a specific binding capacity for damaged DNA and stimulates the ATPase activity of TFIIH 306, 307. Wakasugi and colleagues recently identified the heterodimeric UV-damaged DNA binding protein (UV-DDB) as an interaction partner of XPA which is able to detect a great variety of DNA lesions 308. DDB obviously binds with high affinity to cyclobutane pyrimidine dimers in vitro and communicates with XPA though an interaction of its DDB2 subunit with amino acids 185 and 226 in XPA 308. In contrast, TC-NER recognizes and repairs DNA lesions in the template strand of genes that are being transcribed. TC-NER is triggered by a damage-induced arrest in RNA polymerase II (RNAPII) elongation followed by a displacement of the stalled RNAPII in conjunction with the Cockayne syndrome A (CSA) and CSB protein 309. The stalled RNAPII, in turn, functions as a DNA lesion detector and mediates the enrolment of NER factors and the subsequent repair of the template strand in active transcription processes 310. Following lesion detection by either TC-NER or GG-NER, both routes merge at the level of TFIIH and continue via shared NER mechanisms as mentioned above (for a review see Marteijn et al., 2014) 311. In a final step, the TFIIH complex recruits the endonucleases XPF and XPG to incise the defective strand (Figure 9). The ensuing single-stranded DNA (ssDNA) gap is subsequently replenished by the DNA synthesis machinery (for a review see Marteijn et al., 2014 311; Cambindo Botto et al., 2018 310; Lee et al., 2019 312).
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.