DNA Repair Mechanisms
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DNA Repair Mechanisms
Cells are constantly under threat from the cytotoxic and mutagenic effects of DNA damaging agents. Environmental DNA-damaging agents include UV light and ionizing radiation, as well as a variety of chemicals encountered in foodstuffs, or as air- and water-borne agents. Endogenous damaging agents include metabolites that can act as alkylating agents and the ROS (Reactive Oxygen Species) that arise during respiration. DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by these exogenous and endogenous agents and exposure to carcinogens and cytotoxic compounds like inhaled cigarette smoke, or incompletely defined dietary factors. Failure to repair DNA lesions may result in blockages of transcription and replication, mutagenesis, and cellular cytotoxicity. In humans, DNA damage is involved in a variety of genetically inherited disorders, in aging, and in carcinogenesis. The major forms of DNA damage include SSB (Single-strand Breaks), DSB (Double-strand Breaks), alteration of bases, hydrolytic depurination, hydrolytic deamination of cytosine and 5-methylcytosine bases, formation of covalent adducts with DNA, and oxidative damage to bases and to the phosphodiester backbone of DNA. The vast majorities of these lesions are repaired by BER (Base Excision Repair), NER (Nucleotide Excision Repair), and MMR (Mismatch Repair) (Ref.1).

BER involves the concerted effort of several repair proteins that recognize and excise specific DNA damages, eventually replacing the damaged moiety with a normal nucleotide. BER has two subpathways, both of which are initiated by the action of a DNA Glycosylase. In humans, six DNA Glycosylases have been identified, and each excise an overlapping subset of spontaneously formed, oxidized, alkylated, or mismatched bases. Such DNA Glycosylases bind specifically to a target base and hydrolyze the N-glycosylic bond, releasing the inappropriate or damaged base while keeping the sugar phosphate backbone of the DNA intact. This cleavage generates an AP (Apyrimidinic/Apurinic) or abasic site (i.e. the site of base loss) in the DNA. The AP site is subsequently processed by APE1 (AP Endonuclease-1, also called HAP1/REF1/APEX) which cleaves the phosphodiester backbone immediately 5 to the AP site, resulting in a 3 hydroxyl group and a transient 5’ dRP (abasic Deoxyribose Phosphate) (Ref.2). Removal of the dRP is accomplished by the action of DNA Pol Beta (Polymerase Beta), which adds one nucleotide to the 3’ end of the nick and removes the dRP moiety via its associated AP lyase activity. A DNA Ligase seals the strand nick, thus restoring the integrity of the DNA. Replacement of the damaged base with a single new nucleotide is referred to as short-patch repair and represents approximately 80-90% of all BER. DNA Pol Beta also interacts with XRCC1, which acts as a scaffold protein by bringing the polymerase and ligase together at the site of repair and stabilizing the complex (Ref.3). In cases where the terminal sugar-phosphate residue is resistant to cleavage by the AP lyase function of DNA Pol Beta, DNA strand displacement occur, involving either DNA Pol Beta or a larger polymerase such as DNA Pol Delta for filling-in of gaps a few nucleotides long (Ref.3). This represents the back-up pathway of BER, termed long-patch repair. PARP (Poly ADP Ribose Polymerase), which binds to and is activated by DNA strand breaks, and the recently identified PNK (Polynucleotide Kinase-3-Phosphatase) is important when BER is initiated from a SSB to protect and trim the ends for repair synthesis. The FEN1 (Flap Endonuclease-1) structure-specific nuclease removes the displaced dRP as part of a "flap" oligonucleotide prior to sealing of the nick by DNA Ligase and the PCNA (Proliferating Cell Nuclear Antigen) protein stimulates these reactions (Ref.4), acting as a scaffold protein in this alternative pathway in a way similar to that of XRCC1 in the main pathway.

NER is perhaps the most flexible of the DNA repair pathways considering the diversity of DNA lesions it acts upon. The most significant of these lesions are pyrimidine dimmers (cyclobutane pyrimidine dimmers and 6-4 photoproducts) caused by the UV component of sunlight. The NER process involves damage recognition, local opening of the DNA duplex around the lesion, dual incision of the damaged DNA strand, gap repair synthesis, and strand ligation. There two distinct forms of NER: GG-NER (Global Genomic-NER), which corrects damage in transcriptionally silent areas of the genome, and TC-NER (Transcription Coupled-NER), which repairs lesions on the actively transcribed strand of the DNA. In GG-NER, the XPC (Xeroderma Pigmentosum Complementation Group-C)/hHR23B (Rad23 homolog B) protein complex is responsible for the initial detection of damaged DNA. Conversely, damage recognition during TC-NER does not require XPC, but rather occurs when the transcription machinery is stalled at the site of injury. The stalled RNA Polymerase complex is then displaced in order to allow the NER proteins to access the damaged DNA. This displacement is aided by the action of the CSA (Cockayne Syndrome-A), CSB proteins and XAB2 (XPA Binding protein-2), as well as other TC-NER-specific factors. The subsequent steps of GG- and TC-NER proceed in an essentially identical manner. XPA and the heterotrimeric RPA (Replication Protein-A) then bind at the site of injury and further aid in damage recognition. Next, the XPB and XPD Helicases, components of the multi-subunit transcription factor TFIIH; unwind the DNA duplex in the immediate vicinity of the lesion. The endonucleases XPG and ERCC1 (Excision Repair Cross-Complementing group-1)/XPF then cleave one strand of the DNA at positions 3 and 5 to the damage, respectively, generating an approximately 30 base oligonucleotide containing the lesion. This oligonucleotide is displaced, making way for gap repair synthesis (performed by DNA Pol Delta/DNA Pol Epsilon, as well as several replication accessory factors). Finally, DNA Ligase seals the nick in the repaired strand, thus completing the NER process (Ref.5). In addition, human cells can repair cross-links between the two DNA strands. Natural psoralen compounds and their chemotherapeutic derivatives generate interstrand cross-links by other drugs used for cancer treatment, and to some extent by ionizing and UV radiation. Repair of such cross-links involves the NER genes, the XRCC2 and XRCC3 recombination genes, SNM1 and DNA Polymerase PolQ. In addition, the sensitivity of cells from individuals with FA (Fanconi’s Anemia) points to a role for the FANC group of genes in cross-link repair. At least three syndromes are associated with inborn defects in NER: XP, CS and TTD (Trichothiodystrophy), all characterized by exquisite sun sensitivity (Ref.6). The prototype repair disorder, XP is involved in sun-induced skin cancer (basal cell carcinoma, squamous cell carcinoma and melanoma), which arises from mutations in one of seven genes (XPA-XPG). CS, caused by mutation in the CSA or CSB genes, is a TC-NER specific disorder in which the physical and neurological developments are impaired, resulting in dwarfism and dysmyelination. TTD is a condition sharing many symptoms with CS, but with the additional hallmarks of brittle hair, nails and scaly skin (Ref.7).

The MMR system is responsible for the post-replicative repair of mismatches and small single stranded DNA loops, and it is critically involved in preventing recombination between homologous DNA sequences. MMR removes nucleotides mispaired by DNA Polymerases and IDLs (Insertion/Deletion Loops) that result from slippage during replication of repetitive sequences or during recombination. Defects in this system dramatically increase mutation rates, fuelling the process of oncogenesis. Four principal steps involved in MMR are: (1) recognition of base-base mismatches and IDLs; (2) recruitment of additional MMR factors; (3) search for a signal that identifies the wrong (newly synthesized) strand, followed by degradation past the mismatch; and (4) resynthesis of the excised tract (Ref.8). The eukaryotic MMR system involves two different heterodimeric complexes of MutS-related proteins, MSH2-MSH3 (known as MutSBeta) and MSH2-MSH6 (known as MutSAlpha), and each has different mispair recognition specificity. Heterodimers MSH2-MSH6 focuses on mismatches and single-base loops, whereas MSH2-MSH3 dimmers (MutSBeta) recognize ILDs (Ref.9). Excision and resynthesis of the nascent strand (containing the mismatch or IDL) is performed by a number of proteins including PCNA, RPA, RFC (Replication Factor-C) Exonuclease-I, DNA Pol Delta/DNA Pol Epsilon, FEN1, and additional factors. MMR has an important role in genetic recombination beyond the repair of mispaired bases by regulating the resolution of Holliday junctions and is involved in cancer especially HNPCC (Hereditary Non-Polyposis Colorectal Carcinoma) that exhibit widespread alterations of poly (A) tracts (Ref.9).

DSBs are the most serious form of DNA damage because they pose problems for transcription, replication, and chromosome segregation. DSBs affect both strands of the DNA duplex and therefore prevent use of the complementary strand as a template for repair. Cells have evolved two distinct pathways of DSB repair, HR (Homologous Recombination) and NHEJ (Non-Homologous End Joining). HR-directed repair corrects DSBs defects in an error-free manner using a mechanism that retrieves genetic information from a homologous, undamaged DNA molecule. The majority of HR-based repair takes place in late S- and G2-phases of the cell cycle when an undamaged sister chromatid is available for use as repair template. The Rad52 epistasis group of proteins, including Rad50, Rad51, Rad52, Rad54, NBS1 (Nijmegen Breakage Syndrome-1) and MRE11 (Meiotic Recombination-11) function in the initial steps of meiotic recombination and are also involved in recombination processes in mitotic cells (Ref.10). The Rad52 protein itself is the initial sensor of the broken DNA ends. Processing of the damaged ends ensues resulting in the production of 3 ssDNA overhangs. The newly generated ssDNA ends are bound by Rad51 to form a nucleoprotein filament. Other proteins including RPA, Rad52, Rad54, BRCA1, BRCA2 (Breast Cancer Susceptibility Proteins) and BARD1 (BRCA1-associated RING domain-1), and several additional Rad51-related proteins serve as accessory factors in filament assembly and subsequent Rad51 activities. The Rad51 nucleoprotein filament searches the undamaged DNA on the sister chromatid for a homologous repair template. Once the homologous DNA has been identified, the damaged DNA strand invades the undamaged DNA duplex in a process referred to as DNA strand exchange. A DNA Polymerase then extends the 3 end of the invading strand and subsequent ligation by DNA Ligase-I yields a heteroduplexed DNA structure. This recombination intermediate is resolved and the precise, error-free correction of the DSB is complete. In the NHEJ, where the two DNA ends are connected without the need for longer stretches of homology, repair of a DSB is error prone and frequently leads to small deletions. NHEJ rejoins the two broken ends directly and generally leads to small deletions of DNA sequence. The activity of the Ku70/Ku80 heterodimeric protein is essential for NHEJ. The Ku heterodimer initiates NHEJ by binding to the free DNA ends and recruiting other NHEJ factors such as DNA-PKcs (DNA-dependent Protein Kinase), XRCC4, XRCC7 and DNA Ligase-IV. In addition to the Ku and Ligase-IV homologs, the Rad50, MRE11 and NBS1 genes are also involved in NHEJ, particularly if the DNA ends require processing before ligation. Unrepaired DSBs can be lethal, whereas misrepaired DSBs can cause chromosomal fragmentation, translocations and deletions. Such lesions are potential inducers of carcinogenesis through activation of proto-oncogenes, inactivation of tumor suppressor genes or loss of heterozygosity (Ref.10).

The simplest of the human DNA repair pathways involves the direct reversal of the highly mutagenic alkylation lesion O6-mG (O6-methylguanine) by the product of the MGMT (O6-Methylguanine DNA Methyltransferase) gene. The O6-mG adduct is generated in low levels by the reaction of cellular catabolites with the Guanine (G) residues in the DNA. Correction of the lesion occurs by direct transfer of the alkyl group on Guanine to a cysteine residue in the active site of MGMT in a "suicide" reaction. The inactivated alkyl-MGMT protein is then degraded in an ATP-dependent Ubiquitin proteolytic pathway (Ref.11). Oxidized pyrimidines, such as Thymine (T) glycol and Cytosine (C) glycol, are excised by the hNTH1 glycosylase. This enzyme is ineffective by itself, and the NER enzyme XPG acts as a cofactor for hNTH1. The most deleterious ROS-induced adducts oxoG (7,8-dihydro-8-oxoguanine) is repaired by hOGG1 (8-Oxoguanine-DNA Glycosylase), which excise the oxoG base, leaving behind an abasic site that is restored to Guanine by the cellular abasic site repair machinery. Two DNA glycosylases TDG (Thymine-DNA Glycosylase) and MBD4 (Methyl-CpG Binding Domain Protein-4) excises Uracil (U) and Thymine specifically at deaminated CpG and 5-methyl-CpG sequences. The recently discovered UNG2 (Uracil-DNA Glycosylase-2) and SMUG1 (single-strand Selective Monofunctional Uracil DNA Glycosylase-1) are associated with DNA replication forks and correct Uracil misincorporated opposite Adenine (A). SMUG1, which is unique to higher eukaryotes, probably removes the Uracil that arises in DNA by deamination of Cytosine (Ref.12). The multistep processes of DNA replication, repair, and recombination also require the excision of nucleotides from DNA 3’ termini. Enzymes containing 3’-to-5’ exonuclease activity, such as TREX1 and TREX2 (Three prime Repair Exonucleases), remove mismatched, modified, fragmented, and normal nucleotides to generate the appropriate 3-prime termini for subsequent steps in the DNA metabolic pathways.

Many of the checkpoint proteins are activated in response to DNA damage, and a number of phosphorylation events in DNA repair mechanism depend on activation of these kinases. The Mec1/Rad3/ATM (Ataxia Telangiectasia-Mutated)/ATR (ATM/Rad3-related) family act early in the checkpoint pathways either as DNA-damage detectors or in close association with such detectors. In mammalian cells, these kinases are activated in a lesion-specific fashion, with ATM being specific for agents that induce DNA-DSB, and ATR, probably responding to UV-induced damage. Additional conserved checkpoint components that function in concert with the Mec1/Rad3/ATM/ATR kinases includes the "checkpoint Rad" proteins Rad1, Rad9, Rad17 (Rad24), Rad26, and Hus1, loss of any one of which confers radiation sensitivity and checkpoint defects. Specifically, Rad1, Rad9 and Hus1 are related to the DNA Polymerase accessory protein PCNA, whereas Rad17 is related to subunits of RFC. During DNA replication RFC functions to load PCNA onto chromatin. Just as PCNA acts by tethering replicative polymerases to their template, Rad1/Rad9/Hus1 form a sliding clamp, which has the dual purpose of checkpoint signaling and recruiting DNA repair enzymes (Ref.12). DNA-damage checkpoints in higher eukaryotes have acquired additional levels of complexity. The tumor suppressor p53 is central to these higher eukaryotic checkpoint controls in DNA damage-induced G1 arrest through transcriptional induction of the cyclin-dependent kinases Chk1 and Chk2, and inhibitor p21/WAF1, which binds to and inhibits G1 cyclin-dependent kinases. Activation of p53 in response to DNA damage and other stresses involves complex posttranslational modification of p53 and its negative modulator MDM2 (Mouse Double Minute-2), which is itself induced by p53 at the transcriptional level. MDM2 targets p53 for proteolysis by the ubiquitin/proteasome pathway, and downregulation of this proteolytic pathway following DNA damage allows p53 accumulation. GADD45 (Growth Arrest and DNA Damage-inducible) is one of several known p53 target genes, which is involved in a variety of growth regulatory mechanisms, including DNA replication and repair, G2/M checkpoint control, and apoptosis through activation of JNK (c-Jun N-terminal Kinase)/SAPK (Stress-Activated Protein Kinase) pathway. It binds to several proteins involved in these processes, including PCNA, p21/WAF1, and CDC2 (Cell Division Cycle-2) (Ref.13).

An increasing number of human hereditary diseases that are characterized by severe developmental problems and/or a predisposition to cancer have been linked to deficiencies in DNA repair. Several other classes of DNA damage exist for which repair has been relatively unexplored. New genes may be identified, for instance, involved in the repair of damage caused by lipid peroxidation. Other uncharacterized forms of DNA damage caused by reactive metabolites and catabolites may be found because the genome is dynamic, and ss-regions are temporarily exposed during DNA replication and gene transcription. Positions that are normally protected by base pairing within the double helical structure are vulnerable to group-specific reagents, creating new classes of lesions. New clinical applications relating to human DNA repair genes are certain to emerge. Genomics approaches such as array technology is being used currently to define any DNA repair genes that may be overexpressed in this context or to reverse the potentially deleterious damage that would otherwise destroy the precious blueprint for life.