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DNA damage response

DNA damage induction

Due to the complex chemical composition of DNA, a perplexing diversity of lesions is continuously induced in our genes, which threatens proper functioning. DNA lesions are derived from three main sources:
  1. Environmental agents such as ubiquitous UV and ionising radiation, as well as numerous natural or man-made genotoxic chemicals;
  2. (By)products of normal cellular metabolism, notably reactive oxygen species (ROS) generated by respiration, lipid peroxidation, endogenous alkylating agents, estrogen and cholesterol metabolites and reactive carbonyl species. These compounds generate a diversity of lesions, e.g. ROS alone generate already several different kinds of single strand breaks and >70 oxidative base- and sugar-products in DNA;
  3. Spontaneous disintegration of chemical bonds in DNA, including hydrolysis of nucleotide residues, inducing abasic sites and deamination of C, A, G or 5me-C.
Estimates of daily single strand breaks and spontaneous base losses in nuclear DNA run up to 104 per cell per day. Together with other spontaneous damage, the total may amount to >104-105 lesions per cell per day. Also inflammation may locally cause high levels of oxidative damage. This indicates the magnitude of the DNA damage problem for higher organisms containing large genomes, including mammals.

Consequences of DNA damage

DNA injury has dramatic short and long term consequences. Acute effects stem from disturbance of DNA metabolism, such as transcription and replication, triggering transient or permanent cell cycle arrest or cell death. Long term effects arise from irreversible mutations, causing loss or alteration of gene function. Depending on the type of lesion, its location, the stage in the cell cycle and type of cell or tissue, the outcome of DNA injury (e.g. 8-oxodG) may be biased towards mutagenesis, which in turn triggers carcinogenesis. Alternatively, DNA damage, such as double strand breaks and interstrand crosslinks, may cause cytotoxic and cytostatic effects, which appears intimately linked with ageing. These two critical outcomes of the DNA damage problem, cancer and ageing-related disorders, determine the medical bearing of genome stability and are of major impact for human health in general.
FIG1

Figure 1. DNA damage, repair mechanisms and consequences. DNA damage can be induced by exogenous genotoxic agents, by (by)products of endogenous metabolism including ROS and spontaneously e.g. due to hydrolysis. Two types of consequences can be distinguished: surviving cells may -after misrepair or after replication of the damaged template- acquire mutations or chromosomal aberrations, which enhance the risk of cancer. Alternatively, damage may interfere with transcription or induce replication arrest which may trigger cell death or cellular senescence and thereby contribute to aging. Damage-induced cell death protects from cancer. Right part, top: common DNA damaging agents; middle: examples of lesions that can be introduced in the DNA double helix by the above agents; bottom: the most relevant DNA repair mechanism responsible for the removal of the indicated lesions.
Left part, top and middle: acute effects of DNA damage on cell cycle progression (leading to transient arrest in the G1, S, G2 and M cell cycle phases) and on various aspects of DNA metabolism (notably interruption of transcription, replication and chromosome segregation), which can induce apoptosis or necrosis; bottom: long term consequences of DNA injury causing permanent changes in the DNA sequence (point mutations affecting single genes or chromosome aberrations which may involve multiple genes) and their biological effects. Abbreviations: MMC: mitomycin C and Cis-Pt: Cis-Platin (both DNA cross-linking agents); (6-4)PP: 6-4 photoproduct and CPD: cyclobutane pyrimidine dimer (both induced by UV light); BER and NER: base and nucleotide excision repair respectively. (modified from (Hoeijmakers Nature 2001, 411:366-374.))

The DNA damage response

Mammals have developed a highly sophisticated machinery of complementary genome-stabilising systems to cope with the fundamental DNA damage problem and limit its deleterious consequences. The elaborate DDR apparatus consists of three main types of components.
  1. The heart of genome protection is comprised of an intricate network of DNA repair mechanisms that are highly conserved and as a whole cover most of the wide range of damages induced in the vital genetic information. At least six major, complimentary, but partly overlapping, multi-step damage repair pathways are known to operate in mammals. A separate major repair system is mismatch repair (MMR), which is chiefly concerned with correction of mispaired bases due to replication errors. The division of tasks between the main repair processes is depicted Figure 1.
  2. To solve the problem of lesions interfering with replication, the DDR machinery has acquired a recently discovered class of translesion DNA polymerases that temporarily take over from the blocked regular replication machinery to allow lesion bypass. However, these aberrant polymerases permit translesion DNA synthesis at the expense of an increased mutation rate. A second pathway allowing error-free bypass of lesions is based on re-initiation of DNA replication downstream of the lesion. The resulting gap is filled in by recombinational replication using the newly synthesized complementary strand as template. This largely unexplored system likely exists in humans and is of considerable relevance for preventing carcinogenesis and for this proposal.
  3. To avoid the deleterious consequences of DNA damage, the genome protection apparatus has inserted built-in mechanisms in the cell cycle machinery to sense genome injury and arrest at specific safe points in G1, S, G2 and M phases to allow repair of lesions that otherwise would be converted into permanent mutations or chromosome aberrations.
In addition to these known genome maintenance pathways, nature has developed a complex machinery to specifically deal with telomeres: repeated DNA sequences at the end of chromosomes, which may be perceived as DNA breaks. Telomeric repeats may be lost with every round of replication, in the absence of de novo addition of repeats by telomerase. Thus, cells that have undergone a large number of divisions (as is the case in premalignant stages of cancer), may enter into a crisis, when telomeric repeats are lost and telomeres turn into double strand breaks. The importance of this DDR problem is apparent from the notion that over 90% of all tumours have (regained) telomerase activity or found other manners to overcome this barrier. In addition, mitochondrial DNA is protected from DNA damage by still relatively poorly characterized repair and recombination systems, which are in part borrowed from some of the nuclear DNA repair processes. In view of the relevance for this proposal the base excision repair pathway incorporating the activity of PARP and the process of double strand break repair by homologous recombination are depicted in Figure 2 and 3 and mechanistically described in more detail in the corresponding legends:
FIG2

Figure 2. Schematic of molecular mechanism of base excision repair and the role of PARP A battery of glycosylases, each dealing with a relatively narrow, partially overlapping spectrum of lesions, feeds into a core repair reaction. Glycosylases cleave the damaged base from the sugar-phosphate backbone (stage I in the figure). The resulting abasic site can also occur spontaneously by hydrolysis. The core BER reaction is initiated by strand incision at the abasic site by the APE1 endonuclease (II). Poly(ADP-ribose) polymerase (PARP), which binds to and is activated by DNA strand breaks, and polynucleotide kinase (PNK) are important when BER is initiated from a SSB to protect and trim the ends for repair synthesis (III). In mammals, the so-called short-patch repair is the dominant mode for the remainder of the reaction. DNA polb performs a one-nucleotide gap-filling reaction (IV) and removes the 5'-terminal baseless sugar residue via its lyase activity (V); this is then followed by sealing of the remaining nick by the XRCC1-ligase3 complex (VI). The XRCC1 scaffold protein interacts with most of the above BER core components and may therefore be instrumental in protein exchange. The long-patch repair mode involves DNA polß, pold/e and proliferating cell nuclear antigen (PCNA) for repair synthesis (2-10 bases) as well as the FEN1 endonuclease to remove the displaced DNA flap and DNA ligase 1 for sealing (VII-IX). The above BER reaction operates across the genome (modified from (Hoeijmakers Nature 2001, 411:366-374.)).
FIG3


Figure 3. Schematic of molecular reaction mechanisms for repair of double strand breaks by homologous recombination and non-homologous end-joining. Scenario for the homologous recombination reaction is depicted in the left panel of the figure. To promote strand invasion into homologous sequences, the RAD50/MRE11/NBS1 complex stimulates resection of both DNA ends to create 3' single-stranded extensions (I). Single strand binding protein RPA facilitates assembly of a RAD51 nucleoprotein filament (II). RAD51 has the ability to exchange the single strand with the same sequence from the intact sister chromatid DNA, probably assisted by RAD52 and 54 proteins. The intact double-stranded copy is used as a template to properly heal the broken ends by DNA synthesis (III). Finally, the so-called Holliday junctions are resolved by resolvases (IV). Homologous recombination involves the simultaneous action of large numbers of the same molecules, which are found to be concentrated in radiation-induced nuclear foci. These also include, the BRCA1 and BRCA2 proteins. BRCA2 is involved in delivery of RAD51 to the DSB. As cells in G1 have no identical sister chromatid for error-free repair, HR is primarily involved in repair of replication-associated DSBs. As an alternative, the end-joining reaction simply links ends of a DSB together, without any template, using the end-binding KU70/80 complex and DNA-PKcs, followed by ligation by XRCC4-Ligase4 (see the right panel, stages V-VII). End joining is the major pathway to repair DSBs throughout the cell cycle and is sometimes associated with gain or loss of a few nucleotides. The cell cycle machinery, including the DNA damage signalling protein kinases ATM and ATR, influences the DSB repair pathway choice (modified from (Hoeijmakers Nature 2001, 411:366-374.)).

The DNA damage response and cancer

The DDR is highly relevant to all aspects of cancer. First, numerous cancer predisposing syndromes are attributed to mutations in genes in the DDR pathways such as TP53 (Tumour suppressor P53, mutated in Li Fraumeni syndrome and mutated in more than 50% of all sporadic cancers), ATM (mutated in Ataxia-Telangiectasia), BRCA1/2 (mutated in 50% of familial breast and ovarian cancer patients), XP-A to -G and XP-V (defective in Xeroderma Pigmentosum (XP)), numerous FANC genes (deficient in patients with Fanconi's Anaemia), etc. Second, DDR is important for the onset of carcinogenesis and thereby also for prevention, since most carcinogens are genotoxic, targeting the DNA in a direct or indirect manner. Third, DDR impinges on the evolution to a malignant tumorigenic state, which is driven by mutations and chromosomal instability. The latter can lead to inactivation of tumour suppressor genes, activation of proto-oncogenes and bypassing telomere attrition. Fourth, DDR mechanisms are also relevant to the effectiveness of classical therapeutic treatments, such as radio- and chemotherapy, because these therapies are strongly based on DNA damage induction, which triggers cell death particularly in proliferating cells. Fifth, the DDR also affects therapy resistance, due to attained genetic or epigenetic alterations that prevent cell death (e.g. by apoptosis), which undermines effective cure in most cancer treatments. Finally, hyper- and hyposensitivity of the normal tissue of patients to these classical anti-cancer modalities may at least in part be caused by inter-individual differences in repair and response systems. Thus, the DDR is central to the cancer problem. Due to the intimate links between DDR and carcinogenesis, most tumours have acquired one or more compromised aspects of the DDR in order to reach the number of oncogenic changes required for malignancy and/or to avert cell death despite ongoing DNA-damage induction. Therefore, assessment of the DDR status in tumours is very valuable not only for prevention, diagnosis and prediction of individual cancer susceptibility, but also for predicting individual response to treatment and for counteracting side effects, including long-term consequences and development of therapy resistance.

PARP inhibitors

An illustration of the power of the approach proposed here is provided by the success of potent inhibitors of the single strand DNA break (SSB) repair protein poly(ADP)ribose polymerase (PARP), such as the oral drug Olaparib pioneered by partners of our consortium. Based on specific defects in the DDR, particularly in homologous recombination, the first of a new class of very promising drugs (for BRCA-deficient breast, ovarian and prostate tumours) was developed, which selectively targets the tumour, while leaving normal cells and tissues in the patient relatively untouched. The specific deficiency in the BRCA1 or 2 genes, as an obligate step in tumorigenesis in familial beast cancer, renders tumour cells exquisitely sensitive to PARP inhibitors, while homologous recombination provides a back-up repair system for the repair of spontaneously induced single strand breaks in normal cells in the body. This is an example of synthetic lethality (See Figure 4 for further explanation). Olaparib and several other PARP inhibitors are now in clinical trials for BRCA-defective tumours and others with remarkable results. In view of the complexity of the DDR and the frequent occurrence of partial redundancy, it is to be expected that more therapeutically exploitable cases of synthetic lethality are to be discovered, which may be applied to specific tumours and patients.
fig 4

Figure 4. Rationale of the synthetic lethality of a BRCA1/2-deficiency in tumours and PARP inhibition. Carriers of germ-line mutations in one allele of the BRCA1 or BRCA2 double strand break (DSB) repair genes have a high risk for breast, ovarian or prostate cancer. Tumours of these patients have lost the remaining wild type allele and are deficient in important branches of homologous recombination (HR) repair of DSBs and interstrand crosslinks. In contrast, normal tissues in patients still retain one wild type copy, which is sufficient to carry out normal DSB repair. An important spontaneous source of DSBs arises when replication encounters SSBs (single strand breaks) and turns them into DSBs. To resolve this problem HR involving BRCA1 and 2 is required for complex DNA template switching and fork regression events (see also figure 3). Different types of SSBs occur spontaneously by the action of ROS at an estimated daily rate of ~104 per cell. The majority of clean breaks are quickly repaired by DNA ligases. However, when the ends need processing PARP is required to attract the BER toolkit. Potent inhibitors of PARP have been identified, which cause much higher levels of persisting SSBs. In normal cells of BRCA1/2 patients (which have still one intact BRCA allele) the problem of persisting SSBs causing DSBs upon replication can still be handled by the HR machinery. However, BRCA-deficient tumour cells miss this back-up repair solution and as a consequence show an exquisite sensitivity to PARP inhibitors, such as Olaparib. This has been employed successfully for targeted cancer therapy without significant side effects (Fong et al, N Engl J Med 2009, 361:123-134).