Double-Strand Break Repair

DNA double-strand breaks emerge at a significantly smaller frequency than single-strand breaks, but provide the greater threat to the well-being of cells. Failure to appropriately deal with such lesions leads to large scale chromosomal aberrations, deletions, translocations, chromosome fusions and partial aneuploidy or cell death ^414-416. DSB can arise as a consequence of IR, chemotherapeutics, ROS and VDJ recombination. Of note, many kinds of DNA lesions described in this chapter result in replication stress, fork collapse and DSB formation if unrepaired ^417,418.

- 24 - Homologous Recombination

The genomic maintenance machinery comprises two basic DSB repair pathways, the accurate homologous recombination (HR) and the more error-prone non-homologous end joining (NHEJ) ^419,420. The HR pathway utilizes the sister chromatid as a template for error-free DSB repair, which is only possible in S- and G2-phase of the cell cycle. Homologous recombination is initiated by extended DNA end-processing (5’-3’) by the MRN complex (MRE11-RAD50-NBS), EXO1 and DNA2. The resulting 3’ ssDNA is rapidly coated by RPA. Assisted by BRCA2 and RAD52, RPA is replaced by RAD51, which searches for homologues regions in the sister chromatid. RAD54 controls strand invasion into the intact double-stranded DNA (dsDNA) and proper pairing with the complementary DNA strand.

Finally, DNA polymerases elongate the ssDNA according to the homologous region. This process can either take place with both processed strand breaks at once, forming double Holliday junctions, or by synthesis-dependent strand annealing (SDSA), a mechanism in which after complex dissolution the elongated 3’ ssDNA is annealed with the processed end of the opposing strand break 414,421,422.

Non-Homologous End Joining

NHEJ comes in two sub-pathways, the more accurate C-NHEJ (classic non-homologous end-joining) and the more deleterious and slow alternative end-joining (A-EJ). C-NHEJ starts with a limited resection of the strand ends by the MRN complex as well as others. The processed ends are then bound by the Ku70/Ku80 sub-units which mediates the recruitment of the DNA-dependent protein kinase catalytic subunit (PKcs). The formation of this trimer stimulates the catalytic activity of DNA-PK, resulting in the phosphorylation of itself and several downstream factors (e.g. RPA, WRN, Artemis). At the end of this process, DNA ligase IV reseals the nick in concert with XRCC4 and XLF.

In this pathway, the strand break can be repaired without the induction of errors, in case the ends join precisely. Since no template is used, DNA end-processing can on the other hand be deleterious. This is even more pronounced in the case of the A-EJ pathway, in which minor homology regions are used to align the two DNA ends. Such microhomologies can be located upwards of the actual strand break, making excessive end-processing essential ⁴¹⁴.

PARP-1 in DNA Double-Strand Repair

The role of PARP-1 in DNA double-strand break repair is still a matter of debate and subject of ongoing research. It has been shown that PARP inhibition significantly sensitized cells to agents inducing DSB, and that the repair of these was hampered ^65,423,424. PARP-1 can bind DSBs which triggers its catalytic activity. Interestingly, also PARP-3, but not PARP-2, was reported to interact with DNA DSBs

40,49,65,407. It is believed, that PARP-1 is stimulating a PAR-dependent recruitment of DSBR factors to sites of DSBs and thus facilitates the repair. This was reported to be the case at stalled replication forks.

PARP-1 binds to these sites, becomes activated and recruits the MRN complex which in turn drives end resection and HR repair of collapsed forks ^406-408. Excessive PARylation on the other hand was shown to inhibit MRE11 activity and hinder HR ⁴²⁵. The role of PARP-1 in replication stress-independent HR is supposed to be rather limited. PARP-1 physically interacts with Ku70/Ku80 and DNA-PKcs and targets the DNA-PK as a substrate for PARylation ^426,427. It was suggested, that PARP-1 promotes HR by inhibition of the C-NHEJ pathway, either by competing for DSBs or by protein modification ^428-432. Additionally, an active role in the facilitation of the A-EJ pathway was suggested, with PARP-1 playing a role as a mediating switch between the two sub-pathways ^432-434.

- 25 - 1.3.9 Nucleotide Excision Repair

The nucleotide excision repair (NER) pathway is a versatile repair machinery involved in the removal of a wide range of different lesions. It can be considered to be unique among the DNA repair mechanisms in respect to the fact that it detects an extreme diversity of DNA damages only with a small set of damage recognition and repair initiators. Among the most common lesions are the UV-light-induced 6-4PPs, CPDs and Dewar lesions, chemically-UV-light-induced intrastrand crosslinks, bulky adducts (e.g. BPDE-DNA or other polycyclic hydrocarbons) and ROS-generated cyclopurines. The underlying feature of all these structurally different DNA damages is the varying degree of DNA kinking and helix distortion. The efficiency of lesion removal is, among other factors, determined by the degree of distortion and thus the initial binding and verification of the lesion site ^12,435.

NER is a multistep process neatly choreographed by the sequential assembly of almost 30 proteins. The single steps involve initial damage recognition, local DNA unwinding and damage verification, dual incision on the damaged strand and removal of the oligonucleotide, resynthesis of DNA and sealing of the nick (Figure 1.9) ^436-438.

Two sub-pathways can initiate the NER machinery, the global-genome NER (GG-NER), responsible for maintenance in the whole genome, and the transcription-coupled NER (TC-NER), involved in the detection and removal of lesion sites in actively transcribed genes ^439,440.

Global-Genome NER

The key factor in GG-NER initiation is the heterodimer XPC-HR23B in concert with centrin 2. This complex detects destabilized DNA pairing, resulting in ssDNA gaps (helix distortions) and binds opposite to the lesion on the intact DNA strand ^441-445. The binding affinity of XPC to lesions sites strongly depends on the degree of helix distortion induced by the damage. While 6-4PPs are rather well detected, the more abundant CPDs are mostly unnoticed und are not bound by XPC-HR23B alone. Such lesions are directly bound by a complex called UV-damaged DNA-binding protein (UV-DDB), comprising of DDB1 and DDB2 ^446-449. Binding of this complex further kinks the DNA at the site of lesion, thus facilitating the recruitment of XPC-HR23B ^449-451. DDB1 and DDB2 are part of the cullin-4A (CULcullin-4A)-regulator of cullins-1 (ROC1) E3 ubiquitin ligase complex [CRL] ⁴⁵². CRL is normally kept in an inactive state by the association with the signalosome COP9. By COP9 dissociation and neddylation of CUL4A, the CRL complex is activated, leading to the ubiquitination of DDB2, marking it for proteasomal degradation, as well as ubiquitination of surrounding histones and XPC. DDB1 acts herein as the mediator between the active site of CRL and the substrate specific protein DDB2 ⁴⁴⁹. Ubiquitination of XPC does not facilitate its degradation but enhances its association with the lesion site, locally unwinding the DNA and recruiting the subsequent factor TFIIH 439,453-455. Of note, detection of bulky adducts by XPC does not require the UV-DDB complex. Here XPC binding is the initial event in GG-NER ⁴⁵⁶.

Transcription-coupled NER

The TC-NER is initiated by the stalling of the RNAP II after encountering of a transcription blocking lesion ⁴³⁹. This recruits the loosely associated transcription elongation factor CSB, which alters the DNA-RNAP II interface by wrapping DNA around itself ⁴⁵⁷. In turn this recruits CSA, additional NER factors and p300 ⁴⁵⁸. Here as well, ubiquitination is used as a coordinating mechanism to promote TC-NER. Upon CSA binding of the CRL, COP9 dissociates, CUL4A is neddylated and the CRL ubiquitinates CSB, marking it for degradation, which is an essential step to recover transcription after UV-irradiation ^12,459. This is counteracted by the ubiquitin-specific-processing protease 7 (USP7), which

- 26 -

is recruited to stalled RNAP II by the UV-stimulated scaffold protein A (UVSSA) ^12,458,460. This stabilizes CSB during TC-NER. For the advancing NER, the removal of the RNAP II is an essential step, since it covers the lesion site. This is accomplished by three modes: RNAPII dissociates from the DNA, it performs CSB-assisted backtracking, or if it is resistant to other modes of removal, it is degraded (coordinated by NEDD4)

12,457,458,461-463.

Preincision Complex and Lesion Removal

After initial damage detection and signaling the two sub-pathways merge.

The next step is the recruitment of TFIIH to lesion sites. TFIIH is a multiprotein complex, involved as well in transcription initiation. Upon UV-irradiation its functionality is switched and it serves as a factor in NER. TFIIH contains two helicases with opposing polarities, XPB and XPD. With these, the DNA double helix is further opened around the lesion, creating about 30 nt long ssDNA ^464,465. Facilitated by XPA, this action also verifies the DNA damage.

If no damage is detected, all previous processes can be reversed and processing of the DNA can be stopped ^466-469. XPA binds 5’ of the lesion, at the transition between dsDNA and ssDNA. It interacts with almost all involved NER factors, orchestrating the correct assembly and activity of the preincision complex.

Simultaneously, the heterotrimer RPA (RPA32,

Figure 1.9: The nucleotide excision repair pathway. NER can be initiated by two different subpathways, the global-genome NER (GG-NER) is mainly controlled via lesion detection by DDB2 and XPC in the whole genome.

Transcription-coupled NER (TC-NER) starts with the stalling of the RNAP II at lesion sites in actively transcriped genes, which is sensed and further signalled by CSA/CSB. TFIIH binds the damage site, unwinds the DNA locally and verifies the damage in concert with XPA. XPA further orchestrates the assembly of the preincision complex and correctly postions the endonucleases. ERCC1-XPF performs the incision 5’ and XPG 3’ of the damage site. DNA polymerases fill the gap and DNA ligases restore DNA integrity. Adapted from ¹².

- 27 -

RPA70, RPA14) binds the displaced ssDNA of the intact strand, protecting it from degradation and guiding the subsequent endonuclease activities 437,466,470,471. XPA is further responsible for the recruitment of the 5’ endonuclease ERCC1-XPF. As soon as the 3’ endonuclease XPG has bound, XPF performs a first incision ^472-474. This is sufficient to initiate gap filling DNA synthesis, using the intact strand as a template. Only then, XPG performs the second cut, enabling the removal of the damage bearing oligonucleotide (25-30 nt). This coordinated fashion of cutting, synthesis, cutting, is likely essential to avoid an extended DDR, mediated by ssDNA coated by RPA 472,475-477. Dependent on the cell cycle phase, different combinations of DNA polymerases and ligases are available. In replicating cells POLE in concert with DNA ligase I refills the gap and seals the nick. In non-replicating cells POLD and POLK in concert with XRCC1-DNA ligase III fulfill the task 437,478,479.

Functional Regulation of the NER

Several mechanisms are known to exist, which facilitate the core NER process. Besides direct ubiquitination of core histones by the CRL complex, PARP-1 becomes activated upon UV-irradiation and facilitates chromatin decondensation via PARylation ⁴⁸⁰. PARP-1 activity has further been shown to promote DNA repair by the PAR-dependent recruitment of the chromatin remodeler ALC1. The recruitment of other chromatin remodelers (SWI/SNF and INO80) and histone acetlytransferases (p300 and GCN5) further supports accessibility of damage sites and the repair of lesions. In TC-NER CSB is the central player in chromatin remodeling processes. On the one hand, it has been shown to remodel chromatin nucleosomes on its own and on the other hand, it supports the recruitment and activity of p300 and the chromatin-remodeler HMGN1 458,480-483.

An emerging role of PARP-1 activity in the functional regulation of the UV-induced NER pathway was discovered. PARP-1 has been shown to physically interact with several factors of this machinery or alter their functionality and localization. DDB2 has been shown to stimulate PARP-1 activity, resulting in chromatin decondensation and the recruitment of the chromatin remodeler ALC1 ^480,483. Further, PARylation of DDB2 stabilized the protein, preventing ubiquitination and proteasomal degradation.

CSB interacts with PARP-1 and PAR and its ATPase activity was reported to be inhibited upon this interaction ⁴⁸⁴. XPC is modified with PAR in a covalent and non-covalent manner ⁴⁸⁵. XPA has been show to interact with PARP-1 as well as its product PAR, but the functional consequences of this interaction are so far poorly understood ⁴⁸⁶. Finally, several studies proofed a UV-induced PARP-1 activity and agree on the overall beneficial influence of PARylation for efficient NER conduction ^487,488. 1.3.10 Xeroderma Pigmentosum, Complementation Group A

XPA is a 273 aa zinc metalloprotein, encoded on chromosome 9 (9q22.3) by six exons. It has a calculated molecular weight of 31 kDa, but migrates in SDS-PAGE at about 42 kDa in two close-by bands. The aberrant migration was explained by a high degree of intrinsic disorder, a Glu-rich region and different reduction statuses and disulfide bonds ^489-491.

Due to the large structural disordered regions at the amino- as well as the carboxy-terminus of the protein, so far only the structure of XPA’s central minimal DNA-binding domain (MBD) was resolved by NMR spectroscopy (M98-F219; PDB: 1DU4, Figure 1.10) ^2,492-494. This structure shows its zinc finger (Cys2-Cys2) as well as a motif forming a positively charged cleft essential for DNA-binding

2,494-499. Just recently, two studies suggested to extend the definition of the MBD of XPA (M98-F219 to DBD: F98-T239), since a contribution to XPA’s DNA-binding affinity was observed by a region C-terminal to the MDB ^3,500. Besides this DNA-binding domain, XPA has been shown to interact with a high number of proteins: XPA [homodimer], RPA32 [aa 29-46], RPA70 [aa 153-176], XPC, DDB2 [aa 185-226], CSB, TFIIH [aa 226-273], ERCC1 [aa 67-80], PCNA [aa 161-170], XAB1 [aa 30-34],

- 28 -

XAB2, ATR [aa 188], SIRT1, HERC2, CEP164 [aa 4-97], MutSβ, p300 and RASSF1A. Further, a nuclear localization sequence was identified in the N-terminus of the protein [aa 26-47] (see Figure 4.14A for domain structure). 498,501-517.

XPA binds undamaged and linear DNA, with a moderate affinity loop structures, and with even higher binding strength UV-irradiated and kinked DNA, three-way and four-way DNA junctions. Binding to such structures is greatly enhanced upon interaction with the RPA subunits ^1,499,518.

XPA is a key factor in NER and essential for both sub-pathways, GG-NER and TC-NER. It is involved in the organization of the preincision complex, facilitates DNA damage verification, serves as a scaffold factor and recruits and provides correct placement of the endonuclease ERCC1-XPF. Deficiency of XPA results in almost complete impairment of the NER pathway (<5 % NER capacity compared to XPA^+/-) ⁵¹⁹.

Other than DDB2 and XPC, expression of XPA is not increased upon UV-irradiation, but it has been shown to be regulated by other means. XPA interacts with ATR upon UV-irradiation and becomes phosphorylated at S196. This modification promotes XPA’s nuclear abundance by two ways.

Phosphorylation promotes XPA’s importin-4α-dependent nuclear import and it stabilizes the protein by disturbing the HERC2-XPA interaction. As a result, XPA is no longer degraded and accumulates in the cell ^517,520. HERC2 is a HECT and RCC1-like domain-containing E3 ubiquitin ligase and is suggested to act on a multitude of proteins such as BRCA1 and XPA, as well as other proteins involved in DNA repair, cell cycle control and replication (>300 potential interactors) 517,521-524. The XPA protein exhibits a short half-life, which is tightly controlled by HERC2. This is highlighted in XPA’s circadian rhythm, in which its protein levels vary by the factor 5-10. Additionally to ubiquitination, XPA can be acetylated at K63 and K67. Upon UV-irradiation SIRT1 interacts with and deacetylates XPA and by this, enhances its affinity to RPA32 and facilitates DNA repair ⁵¹⁵. Finally, Pleschke et al. identified a PBM in XPA, located at the C-terminal site of the MDB, partially overlapping with the TFIIH- and DDB2-interaction sites ¹⁴⁷. Interestingly, XPA interacts with PAR in a chain length-dependent manner, with a strong preference for long and branched polymer ¹⁵². Two natural polymorphisms of XPA exist with amino acid alterations in the PBM (R228Q and V234L), both showing improved survival and DNA repair rates upon BPDE exposure as compared to the wild type (wt) variant. Of note, improved survival rates were lesion type specific, since the same XPA variants showed only marginal differences upon UV-irradiation ⁵²⁵.

Figure 1.10: Structural model of XPA’s DNA-binding domain. The dashed line encircles a predicted structure C-terminal of the minimal DNA-binding site. This region includes XPA’s PBM (aa 210-237) and was recently suggested to contribute to XPA’s DNA-binding affinity. The remaining structure shows the previously identified minimal DNA-binding site of XPA (NMR spectroscopy, PDB: 1DU4). Lysine residues predicted to be relevant for DNA-binding are highlighted in blue. Adapted from ³.

- 29 - 1.3.11 NER Associated Disorders

Deficiencies in the nucleotide excision repair pathway are associated with several rare hereditary disorders with a wide range of clinical outcomes, such as significantly increased cancer incidences, developmental disorders, neurodegeneration and segmental progeria. Symptoms vary in both, severity and phenotype for mutations in different proteins, different mutations in the same proteins and even different null mutations in the same proteins ^526-528.

The 13 core NER proteins (XPA-D, XPF/G, CSA, CSB, DDB1, DDB2, ERCC1, TTDA and UVSSA) as well as the NER-associated TLS XPV (POLH) give rise to as many as eight disorders, with overlapping as well as exclusive phenotypes: Xeroderma pigmentosum (XP), XP with neurological complications (De Sanctis-Cacchione syndrome), XP variant, Cockayne syndrome (CS), cerebro-oculo-facio-skeletal (COFS) syndrome, mild ultraviolet syndrome (UVSS), trichothiodystrophie (TTD), and patients with combined symptoms of XP and CS or TTD (XP/CS and XP/TTD) ^528,529. The three superordinate autosomal-recessive disorders XP, CS and TTD can roughly be assigned to the sub-pathways of NER.

Xeroderma Pigmentosum

XP is a rare autosomal-recessive disorder with a frequency of occurrence of about 2.3 per million in Western Europe, provoked by defects in one of the XP genes (XPA-XPV, DDB1/2). It is associated with clinical features ranging from severe sunburn, blistering of the skin and extensive freckling upon UV-light exposure. Incidences of skin and mucous membrane cancer (including squamous and basal cell carcinoma and melanomas) are dramatically increased in patients under 20 years (>2,000 fold of normal population). Additionally, also internal cancer rates are increased. This can be accompanied by progressive neurological degeneration, which is often correlated, but not determined, by defects in XPA, XPB, XPD and XPG, with an onset in the second decade of life. The disease starts early in life (median 1-2 yrs of age) and rapidly progresses with exposure to sun light. Life span is reduced and correlates with sun exposure, and thus cancer and neurological degeneration progression ^527-533. Cockayne Syndrome

CS is caused by defects in CS proteins (CSA and CSB) as well as UVSSA. This syndrome is primarily a developmental and neurological disorder and is not accompanied by increased cancer rates, but with the failure to recover transcription after DNA damage. CS is divided into three forms. CS I is the classical form of CS, with severe neurodegeneration in the first years of life, often resulting in death during the second decade of life. CS II (COFS syndrome) is the most severe form of CS, associated with postnatal retardation of growth and development disorders as well as severe neurodegeneration (cachetic dwarfism, retinopathy, microcephaly, deafness, neural defects, demyelination, ganglial calcification). CS III is the mildest form of CS. Due to its dramatically reduced average life span (12 yrs of age) CS is considered a segmental progeria syndrome 528,534-537.

Trichothiodystrophie

TTD results from defects in the multicomplex protein TFIIH (XPB, XPD and TTDA). It is predominantly associated with developmental defects but not increased cancer incidences. In some patients a severe photosensitivity can be observed. It is characterized by brittle, sulphor-deficient hair and ichthyosis, severe developmental defects, an unusual facial appearance, mild to severe mental

TTD results from defects in the multicomplex protein TFIIH (XPB, XPD and TTDA). It is predominantly associated with developmental defects but not increased cancer incidences. In some patients a severe photosensitivity can be observed. It is characterized by brittle, sulphor-deficient hair and ichthyosis, severe developmental defects, an unusual facial appearance, mild to severe mental