Dna Repair And Human Disease Biology Essay

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The genetic information for every cell in our body is encoded within our DNA, therefore its integrity and stability is essential for life. However DNA damage is unavoidable. The genome is under constant attack from both endogenous and exogenous agents. Exogenous agents such as tobacco smoke are known to cause mutations in lung cells, which can subsequently develop into cancerous cells. UV radiation from the sun/sunbed is also widely known to contribute to the formation of skin cancer as the UV radiation causes distortions in the DNA double helix thereby interfering with the correct replication and transcription of the genome.

Endogenously generated agents e.g reactive oxygen species (ROS) are major source of DNA damage (6)(7) (Kerzendorfer, 2009). ROS species e.g hydroxyl radical, hydrogen peroxide, superoxide, are generated as by-products of normal oxidative metabolism and can directly or indirectly damage DNA. For example, ROS induced lipid peroxidation generates DNA cross linking agents such as malondialdehyde, and since FA patients are hypersensitive to killing by DNA cross-linking agents, these products may be responsible for the congenital clinical features of the disease (Kerzendorfer, 2009).

In addition to these environmental and chemical agents, mutations can also arise during replication as the DNA polymerase can occasionally insert the incorrect nucleotide into the newly synthesised strand. It has been estimated that an individual cell can experience up to one million DNA changes per day (Lodish et al., 2005). If these mutations are left uncorrected, it can result in the loss or incorrect transmission of genetic information which can cause developmental abnormalities and tumourigenisis.

Therefore our bodies have a complex system in place to detect and repair these lesions. This response and repair pathway is closely coupled with the cell cycle checkpoint machinery to limit the adverse effect of these agents on the genetic material. Cell cycle checkpoints represent the restriction sites between each phase of the cell cycle, thus they can stalled or halted the cell cycle to coordinate DNA repair pathways with cell-cycle transitions (Kerzendorfer, 2009) (Branzei D. &., 2006) (Bartek, 2007 ).

By delaying /stalling the cell cycle, allows the DNA damage to be repaired or induces apoptosis if the damage is to severe. Failure to arrest cell cycle when the DNA is damaged can result in mutation fixation, translocations and the gain or loss of genetic material. There are four main ways in which DNA damage can be repaired in mammalian cells -base excision repair, nucleotide excision repair, mismatch repair, homologous recombination, and non homologous end joining. For the purpose of this review, I will briefly describe the pathways involved in each system and then discuss any known defects associated with the repair proteins.

BER is the predominant mechanism that repairs spontaneous point mutations, caused by free radicals and other reactive species generated by metabolism. These endogenous agents interact with the bases causing them to become oxidized, alkylated or hydrolyzed (Clancy, 2008). If the mutated bases are not restored by the excision-repair mechanism, it will lead to permanent change in sequence following DNA replication as one of the daughter strands will contain an incorrect base pair.

The BER system is initiated by DNA gylcosylases as it removes the modified base out of the helix by cleaving the N-glycosidic bond, thus leaving an AP (apurinic/apyrimidinic) site. Mammalian cells contain 11 different glycosylases, each with a specialized function, as reviewed by Barnes and Lindahl (2). E.g UNG, SMUG1, TDG and MBD4 DNA glycosylases can all remove uracil from DNA but in different ways.

UNG is associated with DNA replication forks and corrects incorrect uracil opposite adenine.

SMUG1, unique to higher eukaryotes,is thought to remove U that arises in DNA by deamination of cytosine.

MBD4 excises U and thymine specifically at deaminated CpG and 5-methyl-CpG sequences,

TDG slowly removes uracil and thymine at GzU and GzT base pairs (Wood, Mitchell, & Lindahl, Human DNA repair genes, 2005)..

Some of these glycolyases are bifunctional, while others are monofunctional. Bifunctional glycolyases possess AP lyase activity and can create a single strand break without an AP endonuclease, however this results in a 3' α,β-unsaturated aldehyde adjacent to a 5' phosphate, which differs from the AP endonuclease cleavage product (Fromme, Banerjee, & Verdine, 2004). Monofunctional glycosylases have no associated lyase activity therefore require apurinic/apyrimidinic endonuclease (APE1/APEX1) to incise the phosphodiester bond at the 5' side of the intact abasic site, leaving a 3'- hydroxyl group and a 5' deoxribose phosphate group flanking the nucleotide gap. APE1 also processes other 3' DNA termini that impede further gap filling or religation. Therefore it has an essential role in the repair of abasic sites. Another endonuclease(APE2) is also encoded in the human genome however is thought to have a very minor role due to embryonic lethality of APE1-/- mice (Wood, mitchell, Sgouros, & Lindahl, 2001).

Following removal of the abasic sites, DNA polymerase β, DNA ligase III, and XRCC1 complete the repair process. The net result is the replacement of a single nucleotide and this is called short-patch base excision repair (Hung, Hall, Brennan, & Boffetta, 2005). A subpathway of base excision repair is the long-patch repair which replaces several nucleotides. This pathway appears to play a crucial role in processing oxidized or reduced apurinic/apyrimidinic sites that are resistant to the apurinic/apyrimidinic lyase activity of DNA polymerase β.

In order to switch the repair to the long-patch base excision repair subpathway, a few more nucleotides are added to the 3' end by polymerase σ/ε, generating a flap containing the 5'-sugar phosphate, This flap is removed by flap endonuclease 1 (FEN1), with proliferating cell nuclear antigen (PCNA) stimulating these reactions and acting as a scaffold protein in a manner similar to that of XRCC1 in the main pathway. DNA ligase I then completes this longer-patch form of repair (Hung, Hall, Brennan, & Boffetta, 2005).

Disease associated with BER

The importance of BER in DNA repair can be seen through the use of mutant animal models and these models enable us to determine the role of the BER proteins. Targeted deletion of many specific DNA glycosylases results in viable mice with no apparent phenotype, however mutations in other BER components, including DNA polymerase b, apurinic or apyrimidinic endonuclease, DNA ligase 1 and Flap endonuclease (Fen1) result in embryonic lethal (Friedberg EC, 2003).

Apurinic or Apyrimidinic Endonuclease.

APE1 null mice are embryonic lethality, therefore heterozygotes animals are studied in order to explore the role of APE1 during development. Haplosufficient Apex+/_ mice are viable and show a 40-50% reduction in BER activity in all tissues (Raffoul JJ, 2004; 279). Interestingly, tissue-specific differences in BER activity are observed, with a 35% decrease in the liver, a 55% increase in the testes, and no significant difference in the brain. It was proposed that the basis for these tissue-specific differences in BER activity might be tissue-specific differences in APE redox activation of the tumour suppressor gene p53.

There are also Several sequence variants of APE1, e.g Asp148Glu, that have been suggested to be associated with hypersensitivy to ionizing radiation (Hu JJ, 2001; 22), however this is a long way off from being associated with cancer as there is plenty of contradicting evidence out there. Studies carried out by hung et al, found no association between Asp148Glu and cancer risk (Hung, Hall, Brennan, & Boffetta, 2005).

X-ray Repair Cross Complementing Group 1 (XRCC1)

Deletion of the XRCC1 also results in embryonic lethality.XRCC1 interacts with DNA pol β, APE1/APEX1, Ogg1, and PCNA and in its absence a substantial decrease in the levels of ligase III has be detected (Cappelli E, 1997; 272) (Fan J, 2004; 32) (Goode EL, 2002;11). Over 60 single nucleotides polymorphorisms have been detected in XRCC1, with Arg280His and Arg399Gln being two of the most studied, Arg280His is located in PCNA region and has been suggested to be associated with higher bleomycin sensitivity , Arg399Gln has also been shown to be associated with higher bleomycin sensitivity aswell as high level of aflatoxin b1-DNa adducts. Based on a study carried out by hung et al, they showed that there was association between 399Gln/399Gln and tobacco with increased risk of tobacco-related cancers among light smokers but decreased risk among heavy smokers, suggesting effect modification by tobacco smoking. (Hung, Hall, Brennan, & Boffetta, 2005).

Folate deficiency also results in a functional BER deficiency by stimulating BER initiation but not up-regulating either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol). This leads to a buildup of toxic repair intermediates in the form of single strand breaks (DC, JJ, J, D, H, & AR, 2004; 279). Chemical induction of folate deficiency, by exposing embryos during organogenesis a dihydrofolate reductase inhibitor, causes malformations but does not modify uracil-DNA glycosylase expression or activity (Vinson RK, 2002; 64).

In addition to nuclear DNA repairs, BER is also involved in Mitochondrial DNA damage response. It has been shown to play an important role in aging and in the pathogenesis of neurodegenerative diseases (V. A. BOHR, 2007),

mtDNA repair is beyond the scope of this review . There are several studies published which explore mtDNA repair, however for the purpose of this, I will not go into any more detail.

Nucleotide Excision Repair

NER is another excision repair pathway. The NER pathway is responsible for removing and repairing DNA adducts, i.e chemically modified bases(64). The most frequent example of NER pathway is the repair of [6-6] pyrimidine dimmers, commonly caused by UV irradiation. Pyrimidine dimers distort the normal shape of DNA locally and can block the function of DNA polymerases1, thereby disrupting the replication and transcription process. The NER pathway is divided into two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). TCR repairs bulky lesions of transcribed genes, whereas global NER repairs lesions irrespective of genome location and cell-cycle phase1. The TCR pathway is the most active NER pathway and this is carried out predominantly during G1 phase of the cell cycle.

The initiation response differs for TCR and GGR. TCR may be initiated by a stalled RNA pol II, where as GGR initiation is more complex. Stalled RNA polymerase II recruits small proteins CSA and CSB which are involved in coupling the RNA pol II transcription arrest at the photoproducts sites to the unwinding event (opening of the DNA helix; recruitment of TFIIH). GGR requires damage recognition factors such as the XPC and XPE DNA binding proteins. XPC forms a complex with human homolog of Rad23 (hHR23B) which detects the earliest form of damage, the complex then serves to stabilize the XPA binding to the damaged site with a high affinity for the [6-4] PP. XPC protein may also be required for the transient nucleosomes unfolding, during NER. XPE can also bind to DNA adducts with an affinity similar to that of XPA and XPC however it has a much less prominent role than either of them. XPE forms a heterodimer of a p48 protein, and this p48 subunit is inducible in a p53 dependent manner in human cells.

Following damage recognition, TFIIH is then recruited to this complex. TFIIH is a large protein with 10 subunits, 5 of these subunits are required for both transcription and NER in eukaryotic cells. TFIIH contains both 3'-5' (XPB) and 5'-3' (XPD) helicases subunits which use energy from the ATP hydrolysis to unwind the DNA duplex. XPA stabilises the unwound region and RPA binds to both XPA and DNA.

Xp-G then binds to the complex and further unwinds and destabilise the helix until a bubble of ~25 bases is formed. XpG endonuclease cleaves on the 3' side of the damage and xp-f-ERCC1 heterodimer cleaves the 5'side of the damaged site. The excision process removes 27-29 bases by cleaving 5nt of the 3'side and 24nt on the 5'side of the lesion. This releases the damaged DNA fragment, which is degraded to mononucleotides. The gap is then filled by DNA polymerase exactly as in DNA replication, and the remaining nick is sealed by the action of PCNA, polσ/ε and DNA ligase I.

The use of shared TFIIH subunits in transcription and DNA repair is thought to explain why DNA damage, in humans, is repaired at a much faster rate in regions where the genome is being actively transcribed (TCR). Since only a small fraction of the genome is transcribed in any one cell in higher eukaryotics, TCR efficiently directs repair efforts to the most critical regions, e.g if RNA polymerase is stalled at DNA adduct lesion, CSB is recruited to the RNA polymerase, thus opening up the DNA helix, the recruitment of TFIIH, followed by XP-G, RPA, XP-F as described above.

Human Disease associated with NER Defects-

Mutations in NER genes are linked to some well known genetic diseases for example xeroderma pigmentosa (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD)(Lehmann, A; 2003).

Xeroderma Pigmentosa

xeroderma pigmentosa is an autosomal recessive disease, that is mainly characterized by severe sun sensitivity that leads to progressive degeneration of sun-exposed regions of the skin and eyes and usually leading to various forms of cutaneous malignancy (melanoma and nonmelanoma) [Kraemer et al., 1989, 1994]. The incidence of sunlight induced skin cancer is increased 1000 fold in xp patients.

XPA, XPD, and XPG patients also develop neurological and developmental abnormalities. For example XPA patients can have sensorineural deafness, reduced nerve conduction, difficulty in walking and microcephaly. In these patients the NER repair system is abolished and therefore there is an accumulation of DNA damage in the neuronal neurons that would normally be repaired by NER. Usually XPA patients have the worst phenotype however Jasper et al identified a patient with a complete deletion of XPF gene and this resulted in displaying the severest phenotype, including liver and kidney insufficiency, retarted growth, & early death. Based on this patient and XPF knock out, it assumed that XPF must have a secondary role to NER, which has yet to be identified as the illness observed are far greater than those observed in XPA patients that have no functional NER system.


NER Subpathway

Role in NER

Deletion Effects




Thought to be involved in damage verification??

Abolish NER

V.severe skin problems& CNS defects, eg deafness, reduced nerve conduction, walking difficuties, microcephaly, developed internal tumours



Components of TFIIH-helicase, transcription intiation

No basal transcription

very rare but can result in xp, ttd cs and cos disease



Damage recognition

Sever protein truncations

Severe skin abnormalities but no neurological problems



Components of TFIIH-helicase, optimises transcription

 No basal transcription

xp and ttd and cs and cos




Mis-sense and truncating mutations

Mild skin problems, no CNS problems & late tumour development



Incision on 5'


Partial deletion in most XPF patients =mild phenotype however total deletion of xpf or ERCC-/- = worst phenotype- liver & kidney damage, runting and death shortly after weaning.

Cockayne Syndrome

Cockayne syndrome (CS) is another disease characterized by defects in NER. Unlike XP, CS is a heterogenous disease. Patients have developmental defects including physical and mental retardation, microcephaly, bird-like face, and long limbs. In CS patients, either the CSA or CSB protein is mutated, thus RNA pol II is unable to overcome blockages in transcription during TCR. CS patients are extremely short in statue and have poor health, malnutrition and ataxia (loss of muscle coordination).


Trichothiodystrophy (TTD) is usually associated with mutations in the XPD gene, a gene which encodes a component of the transcription factor TFIIH. The unique characteristic feature associated with TTD is brittle sulphur- deficient hair. Small stature, mental retardation and unusual facial features are also seen in TTD patients.

Double stranded break repair

Double stranded DNA breaks are one of the most severe forms of DNA damage because of their potential to cause gross chromosomal aberrations, often linked to cell death or cancer (Hopfner, 2009)

They are commonly caused by ionizing radiation, cancer drugs and naturally occurring reactive oxygen molecules (lodish et al, (Hales, 2005). Once DSB are detected, the cell initates two response- 1. Checkpoint signaling cascade and 2. DSB repair pathway.

Signal Transduction Cascade

The main players in the signaling transduction response are phosphoinositol-3-kinase-like protein kinase's -ATM- ataxia telangiectasia mutated and ATR (ataxia telangiectasia and RAD3-related). ATM and ATR together with DNA-PKcs can trigger the checkpoint response through activation of the effector kinase CHK1 and CHK2 respectively, which amplify the signal. The activities of ATM and ATR are controlled by interactions between several mediators proteins and various components of the DDR pathway, eg. ATM activity can be influenced by MDC1 and BRCA1 proteins.

When a DSB persists, the DSB ends are detected by MRN complex which is made up of Mre11/Rad50/Nbs1. This complex has multiple functions as it is involved in DSB repair and telomere maintenance, in addition to its role in DNA damage signalling. The MRN complex is the first to recognize a DSB and it recruits ATM thus initiating checkpoint activation.

ATM phosphorlyates and activates chk2 which subsequently phosphorylates Cdc25A resulting in rapid ubiquitin- mediated degradation. As a result Cdc25A is able to remove the inhibitory phosphates of Cdk-cyclin complexes, Cdk1- Cyclin B(G2-M) and Cdk2- Cyclin E (G1-S), thus causing the cell cycle to remain stalled/arrest. In addition to this process, ATM also phosphorylates and stabilizes p53, a tumour suppressor gene, in response to DSB. Phosphorylated P53 induces p21 expression, which inhibits cyclin-CDK2 or -CDK4 complexes, and thus regulates cell cycle progression at G1 phase. See diagram for basic overview. In short ATM is involved in recognizing and signaling unprocessed DSB, -activating the DNA checkpoint.

ATR is activated by the formation of single stranded DNA. ATR exists as a heterodimer with ATRIP and is recruited to ssDNA by ssDNA-binding factor RPA. In order for full activation ATR also requires recruitment of RAD9-RAD1-HUS1 complex, and replication factor C like complex that consists of RAD17 in association with replication proteins rfc2-5. All these complex are phosphorlyated by ATR, and they play a role in helping ATR phosphorlate downstream substrates e.g. Brca1 and p53. The key effector kinase of ATR is Chk1, which functions in a similar manner as Chk2, activating many of the same signaling pathways as Chk2, eg G2/M. however ATR has some unique properties that are not shared by ATM. Loss of ATR results in DNA fragile site expression, DFS are unstable genomic regions that display breaks when under replication stress. DFS tend to be relatively AT-rich and more flexible than non- fragile site regions, which is perhaps why they are so unstable. They are hot spots for sister chromatin exchange and are also thought to play a role in gene amplification events via a breakage-fusion-bridge. In addition to ATR, maintenance of DFS stability also requires BRCA1, SMC1, WRN helicase, CHk1 and the Fanconi anaemia pathways components FANC-A, FANC-B, and FANC-D2. Loss of these proteins/expression of DFS is associated with many cancers.

Upon activation of the checkpoint signaling cascade, DNA damage response is stimulated. However it should be noted that DNA damage response can occur without activating the checkpoint mechanism; only when several DSB are present at the same time or a persistent DSB occurs is the checkpoint signaling cascade activated.

Checkpoint transducers are responsible for phosphorylating the C-terminal tail of the H2AX histones(γH2AX) . γH2AX interacts with MDC1, while MDC1 is simultaneously interacting with ATM and MRN. This interaction results further spreading of γH2AX onto larger chromatin domains on either side of the DSB, and thus plays an important role in stabilizing the accumulation of the MDC1, MRN, ATM and other proteins involved in the DSB response.

DSB repair pathway

The DSBs can be repaired by two major pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Non homologous end-joing DSB repair is error prone method of repair as many sequence errors arise from NHEJ. It occurs mainly during the G1/G0 and early S phase. NHEJ can ligate two end of a DSB together regardless of their sequence homology and chromosomal origin. However movement of DNA within the protein dense is fairly minimal, therefore the correct ends are usually rejoined together, however a couple of base pairs are usually lost in the repair process. Occasionsly broken ends from different chromosomes are joined together, leading to translocation of pieces of DNA from one chromosome to another. Such translocation may generate chimeric genes that can have drastic effects on normal cell function, such as uncontrolled cell growth resulting in cancer formation.

Homologous recombination uses an undamaged sister chromatid as a template for new DNA synthesis (Hales, 2005). It is a highly accurate method of repair and is considered to be error free as no genomic information is lost. However it is limited to the S and G2 phases of the cell cycle as this is when sister chromatid are available (Hales, 2005).

Homologous recombination involves 3 main steps: resection of 5'end DSB, strand invasion & strand exchange and lastly resolution of DNA intermediates.

The 5' end is resected, generating a long 3'single stranded end. The exact mechanisms of how the 5' end is resected remains to be eluted, however the MRN complex and the restriction nucleases are known CtIP, and EXO1 to be involved. Following this, The newly generated ssDNA ends are bound by RAD51 to form a nucleoprotein filament which enable strand invasion into a homologous DNA sequence. In order for RAD51 to assemble onto the ssDNA, it requires assistance from RPA and BRCA2. RPA binds to ssDNA ends with high affinity and is thought to remove its secondary structures. RPA then interacts with BRCA2, and BRCA2 is thought to enable the loading of rad51 onto ssDNA by removing the RPA molecules. Several additional RAD51-related proteins serve as accessory factors in filament assembly and subsequent RAD51 activities however their exact roles in this process are unclear.

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 invasion and D loop formation.

From this point of recombination, several HR pathways can occur, all of which will result in the replacement of the sequence around the DSB by the homologous sequence used for repair.

In the SDSA pathway, one strand will invade and form a D-loop. The 3' end of the invading strand is then extend by a DNA polymerase and will then extend the D- loop allowing recovery of the lost DNA sequences at the DSB. Displacement of the elongated DSB end out of the D-loop allows the possibility of re-annealing DSB ends together using the newly synthesised complementary region. Sequences not involved in annealing are removed and repair can be completed by gap filling synthesis and ligation. This pathway avoids crossovers by doing so appears to account for the genome stability in mitotic cells. RecQ helicases, e.g BLM appear to promote SDSA pathway by removing RAD51 from ssDNA.

An alternative to this pathway is the DSBR pathway, in this pathway the 3' end of the invading DNA strand is extended by DNA polymerase and the parental strands are displaced as growing D loop. The DNA synthesis extends until the displaced single strand matches/base pairs with the other 3 single strand. DNA polymerase then extend the 3' end of the invading strand using the displace parental strand as template. Ligation of the ends results in the formation of two four branched DNA structures called Holliday junctions. This pathway depending on the cleavage can results in crossing over or non crossing over products.

Human Disorders associated with DNA damage signalling

A-T, and NBS, and SS

Human Disease associated with DSB

Blooms disease, fanconi anemia, breast cancer/ ovarian cancer(BRCA1&2)