Genotoxic and non-genotoxic insults arising from endogenous and exogenous sources continually challenge genome integrity. Over 10,000 estimated DNA damaging insults are thought to impose on each human cell per year (Lindahl and Barnes, 2000). These can impinge upon all normal cellular functional aspects with severe consequence to cell homeostasis. Transcription and replication may affect cell viability, whilst DNA lesions arising from DNA damage may induce mutations contributing to aging or diseases such as cancer (Lindhall and Barnes, 2000). DNA repair mechanisms are pivotal for reversal and elimination of intrinsic and extrinsic damage. The DNA damage response (DDR) is a controlled cascade of signal transduction events that has evolved to maintain genomic integrity and cell survival during replication by DNA damage sensing and inducement of various repair factors (Harper and Elledge, 2007). This response can exert its effect via several different mechanisms, dependant on damage severity and type. For example; transcriptional activation, inducing cell cycle arrest, senescence for DNA repair prior to replication, or apoptosis arising from irreparable or severe damage (Zhou and Elledge, 2000). The importance of this and a fully functional DDR are underscored by several diseases, in particular cancer or neurological pathologies such as ataxia telangiectasia from mutated ATM, and Fanconi anemia as a result from mutations in 5 genes which regulate removal of inter-strand DNA cross links mainly through homologous repair and non-end joining pathways. Briefly, at the core of this response are ATM and ATR DDR kinases, activated by DNA damage of dsDNA and ssDNA breaks by interaction with regulators MRN and ATRIP complexes respectively. Activation of these kinases induces a signalling cascade by phosphorylation of a plethora of downstream responsive genes including the well characterised CHK1 and CHK2 and later BRCA1 / 2. Matsukoa et al. (2007) has described over 900 phosphorylation sites of over 700 ATM/ATR responsive
proteins phosphorylated from the DDR on consensus sites by high throughput
proteomic analysis. Other well defined DDR regulators include CDK and DNA-PK. Five major types of DNA repair mechanisms exist and are discussed briefly These events are simplified and illustrated in Figures 4.1.8. - 4.1.9.
Base excision repair
BER is responsible for repair of damaged DNA base modifications, which are the most common form of endogenous damage (Kim and Wilson, 2012). Typical insults include excising of incorrect, damaged bases from deamination (cytosine and 5-
methylcytosine) which results in uracil and thymine mispaired with guanine,
respectively, and C-G --> T-A mutation, or oxidation (8-oxoG). 8-oxoG tends to pair with adenine, resulting in G-C --> T-A mutation. Two pathways constitute this mechanism: short and long patch being repair of a single or more than two nucleotide bases respectively (Robertson et al.2009).
Mismatch repair
This repair mechanism (MMR), in addition to mismatched bases also involves post replication repair of incorrect intercalated bases from error prone activities of DNA polymerases such as slippage during repair or incorrect base recognition. Elevated and mutation prone phenotypes are observed in MMR deficient cells, from MMR
inactivation, whilst germline MMR mutations are cancer prone, in particular for non- polyposis cancer (Zhang et al.2005). Furthermore, MMR is pivotal to many anti-tumour processes such as DNA damaged induced apoptosis (Wu, 1999). The MMR pathway comprises three major steps; 1) recognition of mis-paired bases, 2) excision of the error-containing strand resulting in a gap, and 3) repair synthesis, by gap filling processes.
Figure 4.1.8. Base excision repair – Short patch and mismatch repair pathways A) Base mutation (red) arising from DNA damage (U>G mismatch for eg with UNG) and mismatch base recognised by b) Mutsa and DNA glycosylase (UNG) induces BER pathway, by recognition of and binding to the base lesion. After base binding (uracil for UNG), the base is flipped out of the helix by bending of the DNA and into catalytic pocket of UNG where it is targeted for a subsequent nucleophilic attack to the N glycosidic bond. In c) an AP site is produced as a result of base release by UNG and for MMR; MutLα nicks the 3' or 5' of the mismatched base on the discontinuous strand. This incision by MutLα requires MutSα, RFC, PCNA, and also ATP (Not shown) d) For BER, this site is further processed by AP-endonuclease, APE-1, which cleaves the phosphate backbone 5’ to the AP site, producing a 3’OH and a 5’deoxyribose-phosphate moiety (5’dRP) and for MMR, the resulting DNA is excised by EXO1 a 5'->3' exonuclease in cooperation with RPA ssDNA-binding protein.The 5’dRP is hydrolysed by PolB who fills in the single nucleotide gap in the 5>3 direction ligated and sealed by DNA ligase III, and supported by XRCC1 a scaffold protein to restore the original base sequence. The position of the helix moves from 90 C angle to around 35 C from a – d. Whilst the resultant error free DNA strand is re synthesised by DNA polymerase δ and ligated by DNA ligase (DP) shown in e of MMR.
Nucleotide excision repair
NER is considered to be the most versatile of all DNA repair mechanisms. This pathway responds to and repairs a plethora of unrelated DNA lesions. It removes lesions which may distort the DNA helix, base pairing interference and also blocks DNA transcription and replication. The most commonly responded to lesions by NER are: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs). These are formed between adjacent pyrimidines, constituting the two major types of UV induced lesions. The other most typical is bulky distorted helix lesions induced by chemical agents (Leiber et al.2010). such as cigarette smoke arising from aromatic hydrocarbons, and inter-strand cross linkage induced by chemo therapeutic agents such as etoposide and cisplatin (de Boer and Hoijemaker, 2000).
The NER program comprises two pathways which differ in the recognition stage and whether DNA is transcribed or not. The global genome repair (GGR) repairs
transcriptional inactive genomic lesions (Reidel et al.2003), whilst transcription coupled repair (TCR) focuses upon actively transcribed DNA lesions (Hanawalt and Spivak, 2008). (Sagasawa et al.2008). The importance of the NER repair system is highlighted by several clinical pathologies linked to recessive defects in NER proteins or pathways. At least eleven mutations out of the total 28 NER genes have been linked with rare genetic diseases comprising at least 8 overlapping phenotypes. Examples include: Cockayene syndrome, xeroderma pigmentosum and trichothiodystrophy. These are linked to photo sensitivity as a result of NER errors which typically respond to sun damage. As such, these individuals are pre disposed to skin cancer (Kraemer et al.2007).
Double stranded DNA breaks
Double stranded DNA breaks (dsDNA) are considered the most damaging lesions to genomic integrity. This may impinge upon critical cellular process such as replication and transcription. Conversely, highly controlled dsDNA breaks are thought to be beneficial in some instances. For example, immune system development and during
meiosis (Maizels et al.2005; Loidl et al. 2016). Different repair mechanisms are employed dependant on type of damage and DNA termini break. dsDNA breaks are induced in response to DNA damage insults such as ionising radiation (IR), which are responded to by ATM triggering a cascade of downstream events. Other sources are aberrant enzymatic or endonuclease activity such as topoisomerase activity, or those endogenous such as reactive oxygen species (ROS). These may promote carcinogenesis by genomic rearrangements (Hoijemakers, 2001). Two different repair mechanisms respond to this type of insult; homologous recombination (HR) and non-homologous DNA end joining (NHEJ). Both mechanisms can respond to dsDNA breaks however, one end termini break frequently observed during replication are repaired by HR typically in S phase of cell division. Two stranded breaks are responded to by the continually cell cycle active NHEJ repair pathway. Both pathways are shown in figure 4.1.9.
HR is induced if the damage is not repaired pre- replication by homologous pairing. HR similar to single strand annealing simply defined is whereby homologous information within a secondary intact DNA duplex of a homologous chromosome serves as template to maintain repair fidelity. The HR pathway is highly conserved. HR only functions during S phase and if sequence homology is apt. For example, at least 100 homologous bp are required for this repair mechanism to operate (Leiber et al.2010). Conversely, NHEJ does not require this specific sequence homology and can function at any given time being active in all cell cycle stages. NHEJ thus, may re-join damaged DNA incorrectly. Indeed, NHEJ error is typically always mutagenic (Van De Bosch, 2002). Translocation is a common example of this process, arising from the incorrect joining of independent chromosomes. This occurs due to two different breaks which may occur on separate chromosomes thus giving rise to two DNA termini at each break (Lieber et al.1998). The importance of this is seen in lymphomas and leukaemia where translocations induce oncogene activation arising from aberrant NHEJ events (Gelb and Mederios, 2009). This highlights the importance of error free joining in maintain
Figure 4.1.9 dsDNA break repair by homologous recombination and non-homologous end joining. HR: Black and red lines represent dsDNA homologous sequence. DNA lesions are recognised by the MRN complex recruited to the lesion (black), nucleoytically processed forming the 3’ ssDNA, bound by RPA-RAD51/52 which subsequently invades homologous intact sequences (blue). DNA strand exchange results in a joint molecule of damaged and non - damaged DNA. Missing sequence information at the lesion is restored via DNA re-synthesis (red). Interconnected molecules are processed (grey arrows) by DNA ligation. NHEJ: Following dsDNA breaks, lesions are sensed by Ku70/80 complex to recruit the DNA-PK. This enhances XRCC4-Ligase-Artemis-XLF recruitment and activity facilitating re-joining, and direct ligation. Sequence homology is not required. dsDNA breaks may be joined accurately, however typically translocations of indels are generated.
MRE11- RAD51-NBN 5’’ 3’ 5’ 3’ DNA Pol Ligase I Resolvases Synthesis Ligation Holliday junction OR RPA RAD53 RAD51 RAD54 5’’ 3’ 3’ 5’’ KU70/86 DNA-OKCs XRCC4- Ligase Repair HR NHEJ
Homology search and sensing Resection Strand invasion and D-loop Lesion sensing DNA-PKCs DNA-PK /artemis complex recruitment and activation Artemis Recruitment of XLF-XRCC4-Ligase IV and activation Repair