Living cells are in constant predicament of exposure to agents that damage their DNA. Intact DNA is essential in living organisms to maintain the integrity of genetic information that is passed from one generation to the next. Therefore, any DNA damage needs to be corrected immediately to forestall other serious problems that may arise as outcomes of the damage. Consequently, living cells devise certain mechanisms that correct and minimize DNA damage. Such mechanisms include base excision repair (BER), non-homologous-end joining (NHEJ), and homologous recombinational repair (HRR). This review looked at three original research articles that focused on various aspects of these DNA repair mechanisms. These included the role of DNA polymerase beta during long patch base excision repair in mammals, the exonuclease activity of Artemis on DNA-PK-dependent processing of DNA ends in NHEJ-catalyzed DSB repair, and disorders that arose from DNA damage and errors in various DNA repair pathways. It was realized that DNA polymerase beta played a significant role in LP BER, in mammals. What was initially thought to be an intrinsic exonuclease activity in Artemis was not a natural occurrence. A lot of information concerning DNA damage and repair was known, but there was still need for further research to discover better cures and improve the management of patients with conditions arising from DNA damage.
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Eukaryotic cells face the relentless predicament of genomic DNA damage. Several factors contribute to DNA damage including ionizing radiations, environmental mutagens, cell metabolism involving oxidative reactions, and even DNA’s own intrinsic biochemical volatility (Tor, Maffini and Lowndes 2004). DNA damage prompts numerous rejoinders from the cell including changed expression of genes, arrest of the cell cycle, and a spur in DNA repair. The sequence of DNA bases is extremely valuable for numerous body functions. Therefore, if such errors are not fixed, they may lead to mutations. Strand breaks and errors in cross-links get in the way of procedures such as replication of DNA, separation of chromosomes, and transcription of genes. An intact DNA is a prerequisite for the integrity of genetic information and, therefore, should be maintained at all costs. Errors such as double strand breakage are so crucial that they can result in cell death.
The aptitude of cells to take care of unprompted or environmentally provoked DNA damage determines the stability of the genome and cell viability. As a result, eukaryotic cells have close watch instruments known as “DNA damage checkpoints.” DNA damage checkpoints keep an eye on the integrity of genomes and manage several features of the damage retort. Such responses include cell cycle sequence, transcription of genes, and patching up of DNA.
A “DNA damage checkpoint” is a signal transduction pathway, which detects the incidence of DNA damage and conveys an indication to downstream effectors to implement the numerous cellular responses to DNA damage (Tor, Maffini and Lowndes 2004). Checkpoints indicate the occurrence of anomalous or partly built cell structures due to factors such as the “cell’s own error-prone internal” mechanism, metabolic atmosphere, or external causes (e.g. carcinogens). Damage checkpoints ease the mutagenicity, cell lethality, and genomic instability that arise from DNA damage. They lessen these adverse effects by holding up the progress of the cell cycle and escalating replication of genes that carry out the repair of DNA. They also intensify the activity of the enzymes involved in DNA repair. A number of damage checkpoints in mammals, in addition, serve as tumour suppressors. Therefore, cancer is often coupled with errors in checkpoint signalling paths.
Several studies try to understand the mechanisms and enzymes involved in this crucial process of DNA repair. For example, Durocher (2009) attempts to figure out the initial steps of DNA damage signalling. He pays attention to the sensing and detection of DNA damage, the role of ATM and ATR enzymes in the process. Recent studies by Sakasai and Tibbetts (2009) also attempt to establish the role of these enzymes (ATM and ATR) in regulation of the cell cycle during DNA damage repair.
This paper looks at base excision repair, NHEJ, and HRR as mechanisms of DNA damage repair. It also looks at human diseases arising from failure of timely correction of DNA damage. It does so through summaries of three original research articles in relation to DNA damage and repair.
The Role of DNA Polymerase BETA in BER
The role of DNA polymerase beta in single nucleotide base excision repair (BER) was known, but there was incomplete understanding of the function of this enzyme in long patch base excision repair (LP BER). In the study model, an apurinic or apyrimidine (AP) site from unprompted loss of base or enzymatic exclusion was handled by AP endonuclease giving rise to an intermediate with 3’-hydroxyl and 5’-deoxyribose phosphate (dRP) at intervals one-nucleotide gap (Asagoshi et al. 2010). DNA glucosylase-mediated extraction of bases was accompanied by AP lyase strand incision producing an intermediate with obstructed 3’-hydroxyl and 5’-phosphate groups at the same interval (one-nucleotide interval). The obstruction at the 3’hydroxyl was eliminated by polynucleotide kinase or AP endonuclease (Asagoshi et al. 2010). Therefore, a single nucleotide (SN) gapped intermediate was produced. BER then continued through two sub-pathways differentiated by the patch dimension of the excision repair (SN BER and long patch BER or the LP BER). The two sub-pathways were observed to operate all at once in the cell extract unless a phase was held back or opposed. In the end, one sub-pathway was chosen over the other. SN BER was regarded as well established hence DNA polymerase beta (Pol beta) sealed the SN void and extracted the 5’-dRP group thereby producing a substrate for the activity of DNA ligase. Repair patches that were more than two nucleotides long were handled by LP BER. A “proliferating cell nuclear antigen (PCNA)-dependent branch” was present in LP BER. At that point, “strand displacement DNA synthesis” produced the multiple-nucleotide repair patch together with a displaced flap that was disconnected by flap endonuclease 1(FEN 1) paving way for the activity of DNA ligase. A PCNA-independent path involving filling of gaps by Pol beta appeared to influence the occurrence of LP BER. Gap-filling synthesis by Pol beta was not properly acknowledged in vivo. Little information was known of the enzymology of LP BER’s branch that was PCNA-dependent. There was a significant requirement for validation of the existing working replica of the PCNA-independent branch of LP BER.
The study concentrated on the utilization of a nucleotide excision repair (NER)-deficient cell background. A strand break-containing BER intermediate and a vast 5’-abrasion was also included in the study, with the knowledge that repair by SN BER was impossible. A Neurospora crasa UV damage endonuclease (UVDE) expression vector was used to transform a xeroderma pigmentosum complementation group A (XPA). The cell line that resulted from the transformation expressed UVDE and was labelled UVDE-XPA. A known BER protein (X-ray repair cross complementing group 1 (XRCC1) was depicted to have been admitted to locations of UV induced DNA damage (Asagoshi et al. 2010). In vivo repair of CPDs was initially scrutinized through immunostaining for a short duration of the repair following irradiation by UV. The outcomes authenticated that expression of UVDE in the NER-deficient cells hastened CPD repair. The study also realized that the 5’ end of the single strand nick blocked by CPD was a suitable substrate for Pol beta strand dislodgement synthesis of DNA.
Materials and Methods
The study utilized gamma-32ATP, gamma-32 ddATP, and gamma-32 ddTTP as the nucleotide bases. The main enzymes used were optikinase, terminal deoxynucleotidyl transferase, AmpliTaq Gold DNA polymerase, and protease inhibitors whereas the organisms that were subjected to the experiment were Saccharomyces pombe yeast.
Cell cultures were used to obtain the various tissues to be investigated such as derivation of human XPA-UVDE cell-line from a patient suffering from XPA. Whole cell extracts (WCEs) were prepared and separated by immunoblotting. An improved chemiluminescence scheme was utilized to identify HRP activity. AmpliTaq Gold DNA polymerase was used to magnify the genomic DNA that was isolated from the cells using sequences from synthetic oligodeoxynucleotides (ODNs). The ODNs having a CPD, 42D and 28D were made using a 5’phospahate. The study utilized “a 42 mer CPD-containing duplex DNA” which was made through annealing with the corresponding strand (Asagoshi et al. 2010). Incubation with optikinase in the presence of [gamma -32P] ATP at 37oC for 30min was used to label the CPD with ODN 42D. The labeled ODN was then annealed with its complementary strand and filtered. The resultant double strand was incubated in WCE after which DNA gel-loading buffer terminated the reaction subsequent to incubation. A denaturing polyacrylamide gel electrophoresis (PAGE) separated the products. The gel was scanned and imaged for the purposes of recording the results. In vitro cell extract based LP BER analysis, was done followed by in vitro Pol beta-mediated DNA synthesis. Thereafter, the in vitro FEN 1 cleavage reaction was performed. The reaction products were analyzed for FEN 1 cleavage and in vitro Pol beta-mediated DNA manufacture.
Results and Discussion
The results indicated that UVDE introduced an SSB exactly 5’ to the CPD and 6-4PP. Pol beta was regarded as a good contender for DNA polymerase involved in the repair patch synthesis stage. That was in view of the recommended function of Pol beta in LP BER. An earlier investigation of repair synthesis in the same system revealed a considerable quantity of “amphidicolin-resistant synthesis activity” that was similar in Pol beta-arbitrated repair (Asagoshi et al. 2010).
Mode of Action of Artemis in NHEJ
DNA DSBs caused by ionizing-radiations in higher eukaryotes were primarily repaired by the non-homologous end-joining pathway (NHEJ). Ku, a protein with numerous dimers containing a rare connection and pillar arrangement had an extremely high affinity for DNA ends and conjoined to the site of the DSB. Thereafter, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was engaged to the location of the break and interrelated with the DNA ends and the Ku heterodimer. A protein with three subunits (DNA-PK) was able to phosphorylate substrates from downstream reactions in the active form of serine protein kinase or threonine protein kinase (Pawelczak and Turchi 2010). Processing of DNA terminated by the removal of basic sites and structural impairment e.g. 5’hydroxyl groups, and 3’phosphoglycolates was needed prior to ligation of the double strand break by XRCC4 and XLF complex. Several enzymes were thought to take part in processing of DNA such as Werner protein, polynucleotide kinase (PNK), FEN-1, DNA polymerase lambda and mu, and Artemis (nuclease).
Outcomes suggesting participation of Artemis in the NHEJ pathway were based on in vivo information, which indicated that cells lacking Artemis were “more sensitive to IR than wild type counterparts.” Artemis displayed DNA-PK dependent endonuclease activity on various DNA strands. Previous studies suggested that endonuclease activity of Artemis was triggered by phosphorylation and binding by DNA-PK instigating a conformational alteration in Artemis’ C-terminal region. These events eliminated autoinhibition of the active site of endonuclease by Artemis. Other studies, on the other hand, implied that autophosphorylation of DNA-PK initiated a conformational alteration in the kinase bound to DNA. The result of these events was a change in the appearance of DNA, which enabled identification and cleavage by Artemis. These two suggestions proposed that the endonuclease activity of Artemis was dependent on ATP and DNA-PK despite their somewhat different machinery. It was also suggested that Artemis had a built-in 5’-3’ DNA-PK-independent exonuclease activity (Pawelczak and Turchi 2010).
Artemis, a unique enzyme with two active sites, belonged to a group of metallo-beta-lactamase fold, which comprised of enzymes that acted on nucleic acids. It had both exonucleas and endonuclease activities although its exonuclease activity in vivo was unknown. Artemis was fractionated in a baculovirus expression system and before the demonstration of the separation of its enzymatic activites. A protocol involving three stages was utilized to biochemically separate Artemis’ exonuclease and endonuclease activities.
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Materials and Methods
The first step involved the cloning, protein expression, and purification of Artemis gene. PCR was used to magnify the gene before cloning. The isolated genes were transfected with recombinant baculovirus that expressed the [His]6-Artemis protein. Protein purification of DNA-PK was performed followed by, SDS-PAGE and western blot analysis. In vitro DNA-PK kinase assays were performed at 37oC followed by in vitro exonuclease assays using 5’ radiolabeled DNA substrate. Finally, in vitro endonuclease assays were carried out using the 5’ radiolabeled hairpin substrate with a 6 base single-strand overhang and the 3’overhang DNA substrates (Pawelczak and Turchi 2010).
The study revealed the expression of [His]6-Artemis protein through Coomassie Blue staining of an SDS denaturing gel. The presence of the protein was established by western blot analysis. DNAPK-dependent phosphorylation of [His]6-Artemis in the cell-free extract was also revealed. The appearance of a prominent band in DS-PAGE of a mixture of DNA-PK, DNA, and [gamma-32P] ATP, and [His]6-Artemis protein revealed that indeed Artemis contained eleven serine or threonine residues that were phosphorylated by DNA-PK in vitro.
It was realized that Artemis did not contain an intrinsic exonuclease as was believed earlier. Mutations in proteins involved in endonuclease activity did not affect the exonuclease activity of Artemis. The different results that were obtained did not also clarify the machinery of Artemis activation. Therefore, there was the need for extra studies to illuminate the role of exonuclease processing in NHEJ and the protein accountable for that activity.
Response in Human DNA Damage and the Associated Disorders
The key role of the cell cycle was to guarantee the loyal replication and conduction of genetic constituents to descendant cells. Cell cycle checkpoints signified the restriction points between each stage of the cell cycle whereby the entire process could be cut short or arrested to assist the suitable sequential synchronization of the process making certain that each stage occurred in an orderly manner, or to give time for the repair of DNA. Studies involving yeast and Xenopus systems revealed lots of data on the machinery of the cell cycle regulation in the repair of DNA damage. Faults in the proficiency of the cell cycle checkpoint resulted in a wide range of human congenital ailments. A careful study of these circumstances enabled the establishment of the effects of cell cycle defects on normal human progress and genetic permanence.
This paper reviewed the interplay between DDR and cell cycle machinery using certain human ailments whose aetiology was emphasized by imperfections in features of both processes (Kerzendorfer and O’Driscoll 2009). The paper also discussed the evidence of functional consequence of haploinsufficiency of DDR components and its association with “Genomic Disorders.” In addition, the study revealed the direct repercussions of current studies on the comprehension of the origin of various clinical traits witnessed in DDR-defective disorders. It ultimately outlined how improved knowledge on the DDR helped precise diagnosis, therapy, and management of patients suffering from these diseases.
The Origin of Endonuclease DNA Damage
Many varieties of DNA damage are caused by the continual exposure of the cell to endogenous and exogenous driving forces. Production of explicit DNA breaks is caused by the impact of DNA replication and transcription folks with these damages (Kerzendorfer and O’Driscoll 2009). Efficient DNA impairment response and repair paths work together with the cell cycle checkpoint mechanism to minimize the unpleasant effects of these agents on genetic makeup. Mutation fixations, translocations and loss of genetic material are caused by the failure of the cell cycle to arrest after DNA damage. These occurrences hinder the safeguarding of genomic stability and subsequently cause malignant transformation. Nearly all of the human conditions caused by faulty response to DNA repair normally show as in-born (congenital) clinical defects, a phenomenon that proposed the bulk of DNA damage from endogenous factors were substantial and could negatively mar human development if not handled appropriately. A precise case was microcephaly, which exhibited diminished head circumference indicating anomalous development of the brain. Microcephaly was coupled with various groups of conditions such as “LIG4 syndrome, Seckel syndrome, Nijmegen breakage syndrome, Fanconi anaemia, Cockayne syndrome and some Xeroderma pigmentosum complementation groups” (Kerzendorfer and O’Driscoll 2009). Reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, and superoxide that were produced as by products of regular oxidative metabolism directly or in some way, harm DNA. Lipid peroxidation influenced by ROS produced DNA cross-linking substances such as malondialdehyde. Therefore, cells from patients with Fanconi anaemia were extremely sensitive to destruction by DNA cross-linking vehicles.
DNA Damage Response Indicators and Cell Cycle Checkpoints
The cells’ rejoinders to DNA damage were divided into signal transduction response and cell response to damage. Signal transduction response included procedures that regulated the cells response to the damage such as cell cycle arrest and apoptosis. Its main components were the phosphoinositol-3-kinase-like protein kinase’s ATM (Ataxia telangiectasia) and ATR (Ataxia telangiectasia and Rad3-related) (Kerzendorfer and O’Driscoll 2009). ATM was set into motion by DNA double strand breaks (DSBs) whereas exposed single stranded segments of DNA (ssDNA) triggered ATR. Both enzymes phosphorylated similar substrates and overlapped in their functions.
Key Human Disorders of Defective DNA Repair
Xeroderma pigmentosum (XP) was recognized as the initial human ailment in the repair of DNA damage due to UV-stimulated photoproducts. XP was revealed to be initiated by mutations in diverse constituents of the nucleotide excision repair (NER) pathway (Kerzendorfer and O’Driscoll 2009). Continued neurological deterioration was also commonly witnessed in XP even though its track was erratic. The neurological deterioration was observed as an anomalous gait, sensorineural deafness, and a deficiency of profound tendon reflexes. A number of XP complementation assemblage also showed structural neurological deformities, for example, cerebellar atrophy, microcephaly, and engorged ventricles.
Cockayne syndrome (CS) and Tricothiodystrophy (TTD) were also human disorders characterized by a defective NER (Kerzendorfer and O’Driscoll 2009). Patients with CS had an extraordinarily short physique together with overall poor health and malnutrition (deep cachexia), a progeroid appearance, and loss of muscle synchronization (ataxia). The most significant neurological mark of CS was continued neuronal demyelination.
Major human syndromes of faulty DNA damage signalling included ataxia telangiectasia (A-T) was a cell cycle check-point disease since cells from those patient exhibited errors in “G1-S, intra-S, and G2-M cell cycle checkpoints” in reply to DSBs. Non-homologous end joining-defective disorders included LIG4 syndrome, Art-SCID, XLF or Cernunnos-SCID. Numerous other disorders due to defects in DNA repair were mentioned including their key features and the exact point within the path that was affected.
Diagnosis and Management of DDR-Defective Disorders
Precise recognition, diagnosis, and categorization of DDR-defective disorders were central to correct care of patients with such conditions. Information on genetic defects at the molecular level was critical in helping genetic counselling and planning of families in the families affected. There was no known cure for DDR-defective ailments. Therefore, their management or therapy was only analgesic. Antioxidant-based treatments were commonly utilized in the mitigation of the continual neurological deterioration in cases such as A-T. Aminoglysoside antibiotics in some A-T cases led to read-through of stop codons and were studied as possible cures for Cystic fibrosis, Hurler’s syndrome, X-linked Retinis pigmentosa, and Diabetis insipidus (Kerzendorfer and O’Driscoll 2009). Bone marrow stem cell transplantation (BMSCT) was the only realistic curative alternative. Tailoring of such treatments was only possible through the depiction of DDR-defective ailments. For example, the realization that Graft-versus-host disease (GvHD) was a likely grave impediment during BMSCT initiated the use of cyclosporine a (CSA), a cyclic undecapeptide characteristically used as a prophylactic medication for GvHD.
It was concluded that the escalating number of human ailments due to flaws in response to and repair of DNA damage ought to give researchers a fundamental insight into the root and inheritance of these disorders, and how the affected paths impacted on usual growth and genetic stability. That primary information could then aid in ensuring better management of affected individuals and even facilitate the creation of more effective therapies for all these diseases.
In the determination of the role of Pol beta in BER, it was confirmed that Pol beta used the nicked substrate that contained CPD for LP BER synthesis of DNA. It was realized that Pol beta exhibited similar activity on the “nicked CPD substrate and the 2-nucleotide nicked CPD-flap substrate” (Asagoshi et al. 2010). FEN 1 activity on those two substrates was also similar to that of Pol beta. However, FEN 1 activity was twenty times higher on the 5-nucleotide nicked CPD-flap substrate, indicating that FEN1 could regulate the size of a repair patch because of its robust activity on the CPD-containing 5-base flap (Asagoshi et al. 2010). The study, therefore, suggested the investigation of the function of Pol beta in mammalian LP BER in a tentative scheme where as strand notch 5’to a CPD laceration enforced mending through the constitutively expressed LP BER sub-pathway.
The mutational studies that were conducted to establish the interaction between Artemis and DNA-PKcs gave differing outcomes. The uncertainty of these results “have left the mechanism of endonuclease activation an open question” (Pawelczack 2010). That statement implied that there was room for further investigation to investigate the actual steps behind the activation of endonuclease activity in Artemis. However, the discovery that DNA-PKcs phosphorylated Artemis before it gained endonuclease activity gave the starting point for future studies. In addition, the discovery of DNA-PKcs phosphorylation mutants brought about the conclusion that automatic phosphorylation of DNA-PKcs was necessary to assist the Artemis-catalyzed endonuclease activity. More information was also obtained from the successful identification of a “sub-set of mutants that functionally abrogated endonuclease activity via upsetting metal coordination” (Pawelczack 2010). The knowledge that metallo-beta-lactamase group enzymes possessed one active site, which was responsible for all enzymatic actions, gave more insight on the probability of finding exonuclease activity in the active sites from the generated mutants.
Although many diseases linked to errors in DNA repair were known, more details on some of the diseases were still missing. For example, it appeared that a number of clear-cut human genetic disorders related to microcephaly and short physique seemed to demonstrate a flawed ATR-dependent DDR. Such an association, although strong, was only an association. For that reason, additional work utilizing complementary systems such as gene targeting or tissue specific knockdown in the murine system was needed to confirm unequivocally the connection between ATR pathway dysfunction, haploinsufficiency and these developmental irregularities (Kerzendorfer and O’Driscoll 2009).
It was also imperative to focus attention on the centrosome as a source of other proteins that functioned in the DDR. A large number of Seckel syndrome contributing genes awaited identification. Therefore, the centrosome characterized a budding source of new DDR disease-causing flaws, and there was the need to determine whether those new Seckel syndrome genes encoded proteins localized in the centrosome or structural centrosomal constituents (Kerzendorfer and O’Driscoll 2009).
Mosaic variegated aneuploidy (MVA) was an inborn defect in the spindle checkpoint, which exhibited clinical and cellular inconsistency giving it genetic heterogeneity. There was a probability that mild defects in other elements of the SAC were contained in MVA. It was essential to investigate whether new MVA causal errors occurred in constituents of the DDR that also influenced SAC function. In conclusion, the field of genetic disorders due to DNA damage repair had vast information that required further investigation.
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