DNA Repair Mechanisms - Coursework Example

DNA REPAIR MECHANISMS DNA Repair Mechanisms Name Course Instructor Institution Date DNA Repair Mechanisms Introduction The realization of the importance of preservation of genetic integrity is one of the major developments within the field of biomedical genetics over the last several years (Budman & Chu, 2005). Alteration of genetic information has major impacts not only on carcinogenesis, but also on ageing and other aspects of human health (Leibeling et. al. 2006). Understanding the detailed mechanisms through which genome integrity is maintained, including DNA damage repair mechanisms, is thus of pivotal importance for improvement of numerous quality of life issues. Mechanisms have evolved to help protect human genetic material from endogenous and exogenous damage that are vital for the survival of a human being or other living organisms (Sinha, & Häder, 2002). This ability to recognize damaged DNA and simultaneously regulate progression of cell cycle and DNA repair is very important for genomic stability, and defects in these pathways are hallmarks of cancer (Schonthal, 2004). The DNA damage response pathway is a multi-component signal transduction network that consists of a multitude of proteins whose functions are still a subject of discussion. This paper focuses on the current findings in the filed including identification and analysis of DNA repair mechanisms that contribute to resistance against nucleoside analogues. There are proofreading mechanisms that operate during DNA replication. Apart from this, the body contains a number of mechanisms; for instance, the skin and melanin to protect from ultraviolet damage and enzymes such as superoxide dismutase that deactivates ROS to protect DNA from damage (Lodish et al. 2004). DNA repair is integral in protecting against mutations that could otherwise lead to cancer. Human body has a number of mechanisms that repair DNA damage. These include base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and double-strand break repair (Plosky et. al. 2002). Base excision repair, mismatch repair, nucleotide excision repair mechanisms all rely on DNA being a double stranded structure with the same information contained in both strands. When the damage affects only one strand, it can be repaired accurately by excision and replacement, with new DNA being synthesized using complementary strand as a template (Wilson & Lieber, 1999). Deoxyribonucleic acid (DNA) is a very active molecule and it highly reacts with other molecules; and in the process of reaction, some damages may occur or take place. There are two types of damages that occur during reaction, endogenous and exogenous. The endogenous reaction involves the reactive oxygen species as the resultant from normal metabolic byproducts that are likely to attack DNA; the exogenous involves all external agents attacking DNA such as ultra violet radiation, high temperatures, cancer chemotherapy, and radiotherapy (Wilson & Lieber, 1999). There are three different stages in DNA repair mechanism, namely; base excision, nucleotide excision, and mismatch repair. In base excision, DNA may be modified by deamination or alkylation. The modified or damaged base is called the abasic site; the DNA glycosylase can recognize the abasic site and take away its base (Crowe, et al. 2006). Then the following process is the removal of the endonuclease abasic site and the adjacent nucleotide. The void remained after the removal of the AP is then filled with the DNA polymerase and the DNA ligase. The other stage involved in the DNA repair mechanism is the nucleotide excision, in this process, UvrA, uVRb and UvrC are involved in the removal of the damaged nucleotide such as the dimer induced by ultraviolet radiation. The gap that is created after the removal is then filled by the DNA polymerase I and the DNS ligase. In yeast, the protein similar to the Ultra violet radiation is called the RADXX for example Radiation 1, Radiation 2 Radiation 3 (Bjorksten, Acharya, Ashman & Wetlaufer, 1971). The other DNA repair mechanism is the Mismatch repair, for this kind of repair to take place, the system has to identify which base is the correct one for the process. The E. coli is only possible in the presents of a special methylase called the Dam methylase; these enzymes can only methylate all the adenines that occur. After the DNA replication, the template strand and the new strand that can be formed can be distinguished. The DNA repair process can only begin with the protein mechanisms called the MutS. This MutS which binds to mismatched the base repair. Then the other mechanism called MutL is recruited to the complex and activates with the other mechanism called the MutH binds to GATC sequence. Activation of the MutH cleaves the unmethylated strands at the GATC site. Alternatively, the segments from the cleavage site to the mismatch site are then removed from the exonuclease (Budman & Chu, 2005). If the cleavage occurs on the other sides of the mismatch, then the exonuclease are used to degrade the single stranded DNA and the DNA. Then the gap is then filled with the DNA polymerase III and the DNA ligase. The damage of DNA leads to activation of cell cycle check points. Activation of checkpoints pauses cell cycle and gives cells enough time to repair. Checkpoint activation is controlled by two chemicals namely kinases, ATM and ATR. The three chemicals perform different and distinct functions. ATR responds to stalled replication forks while ATM responds to DNA double strands break and disruption in the chromatin structures. Checkpoint activation is controlled by some proteins. They include BRCA 1, MDC1 and 53PB 1 (Ataian and Krebs, 2006). These proteins are a necessity in since they relay checkpoint activation signal to downstream proteins. When the DNA damages exceed the capacity of cells to be repaired, the accumulation of error results in early senescence, apoptosis or cancer (Branze & Foiani, 2008). It can also lead to inherited disease associated with faulty DNA repair function. This results to premature ageing of a person and increased cancer risk. MRN Complex The MRN complex is a protein complex in the human body consisting of Mre 11, Rad 20 and Nbs 1 also known as Nibrin in the human body and Xrs2 in Yeast. The MRN is linked to many DNA metabolic activities including DNA double strand breaking. The DNA within the human body is continually exposed to DNA damaging agents. These damaging agents include ultraviolet light, mutagenic chemicals and reactive oxygen species (Leibeling et. al. 2006). DNA double strand breaking is a form of DNA damage that is inflicted by mutagens. Double strand breaking is generated when two complementary strands of the DNA helix are broken simultaneously, and the two are close to one another. DNA double strand breaking if not repaired correctly may lead to severe consequences. One of them is the possibility of occurrence of chromosomal instability. Double Strand breaking may be induced by a number of factors; they include ionizing radiation, reactive chemical species, and normal cellular process one of which is cell replication (Budman & Chu, 2005). On detection of Double strand breaking in the cells, DNA repair proteins are recruited to the site of the strand breaking. DSB pathway is then activated, and induction of DSB repair and cell cycle arrest and activation of checkpoint occurs (Budman & Chu, 2005). One of the ways to repair DSB that occurs in the G2 phase of the cell cycle is Homologous Recombination (HR). The diagram above represents a model explaining the role for MRN and ATR in maintaining proper replication (Sinha, & Häder, 2002). Homologous Recombination starts with the recruitment of the MRN repair complex to the DSB site. Once at the site, MRN activates ATM kinase and tethers DNA ends. ATM in turn, initiates signaling cascades, which leads to, resection of the DSB to produce single stranded DNA. The resulting single stranded DNA act as a substrate to Rad 51 (Sinha, & Häder, 2002). The single- stranded binding DNA (SSB) protein family plays a vital role in the repair of the DNA, in all domains of life. The ordinary single- stranded binding protein and the replica protein A are some of the members of the single- stranded protein family. The replica protein A plays a fundamental role in both DNA replication DNA repair pathway. Other members of the single-stranded binding family Hssb1 and Hssb2. HSSB1 and Hssb2 are both structurally more similar to bacteria and archaeal SSB than the replication protein A (Sinha, & Häder, 2002). Hssb1 is essential for efficient signaling of Double strand breaking after exposure to ionizing radiation. HSSB1 is also essential and is crucial for efficient recruitment of MRN complex and subsequent downstream patterns. HSSB1 form a DNA damage independent complex with Mre11 Rad50. The Mre11 protein has the capability to bind DNA; it possesses both exonuclease and endonuclease activities. It acts as the bridgeNBS1 and RAD50. RAD50 is characterized by its long coiled-coil arms. It uses its long arms to hold the end of broken arms together. The NBS1 has no enzymatic but acts as a mediator in protein- protein interaction and helps the MRN complex in performing its many functions. NBS1 interacts with ATM kinase, the MDC1 adaptor protein, and the CtlP a tumour suppressor protein that promotes the processing of the DSBs by MRN (Budman & Chu, 2005). DSB activates the ATR signaling the homologous recombination repair. The core function of MRN complex is to prevent excessive DNA breakage and genomotic instability and hence prevent the genetic diseases like cancer. An experiment carried out on breast cancer patients clearly indicate that, there are significantly high enhanced incidences of MRN eberrant tumors, among the familial breast cancer linked to carriers of BRCA1 and BRCA2 mutations as compared to all non carriers of the mutations (Budman & Chu, 2005). Opoisomerase Inhibitors Topoisomerase inhibitors are enzymes that regulate the winding of the DNA helical structure. Enzyme Topoisomerase regulates both the over winding and under winding of the DNA. There are numerous types of topoisomerase inhibitors each specializing in different types of DNA manipulation. Topoimerase 1 make single-stranded breaks allow the process unwinding in order for transcription or replication to take place, topoisomerase 1 also prevents the overwound or underwound of DNA helix during transcription or during replication. Under-winding or overwinding if left unchecked has potential of impeding the progress of the protein machinery involved in the process taking place. Topoisomerase 111 is essential for introduction of single strand breaks required for DNA swapping of matching sister chromosomes in order to shuffle genetic information. There are two classes of DNA topoisomerase. They have been classified in accordance to their catalytic mechanisms. Type 1 topoisomerases do not require ATP; they act by forming a transient single-strand break through which they achieve relaxation. Type 11 enzymes are usually ATP dependants’ dimeric enzymes. They are able to make double strand break hence creating a DNA- linked protein gate allowing duplex passes. Both type 1 and 11 enzymes are efficient in relaxing supercoiled DNA helix during transcription or replication. The diagram below represents a topoisomerase inhibitor that unsilences the paternal allele of Ube3a in neurons. Both topoisomerases 1 and 11 function during replication to avert tension generated as a result of replication. However, Topoimerase 1 has a role to play during transcription. During transcription, topoisomerase 1 is usually deployed to the transcriptional active regions (Plosky et. al. 2002). Topoisomerase 11 has a role to play during segregation of daughter chromatin after completion of DNA replication and the processes of chromatin condensation and mitotic segregation. Type 11 topoisomerases are ubiquitous enzymes. They can be divided into three distinctive but related clusters based on amino acid sequence and homology (Plosky et. al. 2002). All type 11 enzymes exhibit multiple activities including DNA binding, DNA cleavage reunion and ATP hydrolysis. Vitro binding studies have proven that, there is a common interaction between eukaryotic topoisomerase 11 with both anineoplastic inhibitors (Schonthal, 2004). Topoisomerase inhibitors affect many aspects of DNA metabolism. It affects replication, recombination, and repair. Epipodophyllotoxin etoposide and the intercalator amsacrine inhibit type 11 topoisomerase by interacting with the ATP binding site (Schonthal, 2004). Type 1 topoisomerase inhibitors produce DNA strand break; camptothecin is an example of type 1 topoisomerase (Schonthal, 2004). There are different types of type 1 topoisomerase inhibitors, and they produce strand breaks at different chromosomal sites. DNA damaging agents, DNA synthesis and topoisomerase inhibitors, have the capability of altering chromosomal structure by various mechanisms. These mechanisms can induce DNA repair functions. Topoisomerase plays indirect role in DNA repair. Change in gyrase or topoisomerase 1 inhibitor activity alter super coiling and thereby alter the expression of DNA repair gene. DNA Gyrase, however, plays a direct role in post replication repair. Powerful genetic systems exist in the lower eukaryotic systems. The genetic system helps to assess the importance of topoisomerase. Researches show contrasting results after separate examination on the repair of UV-induced damage in CHO cells in two contexts. Ultimately, Repair in a transcribed gene, dihydrofolate reductase {DHFR}, was unaffected when cells were treated either with a combination of camptothecin and a topoisomerase 11 inhibitor while in genome wide repair was diminished by all three inhibitors (Wilson & Lieber, 1999). The outcome was that there’s a minimal effect on Genosome wide repair by topoisomerase inhibitors. However, No inhibition occurred when only a single inhibitor was used in the DHFR. Borrowing from this research, it is only quite evident that either topoisomerase 1 or 2 is a requirement for gene-specific repair and that inhibition of either enzyme alone is insufficient to inhibit, repair and as such, inhibition of both enzymes affects repair of UV damage significantly (Wilson & Lieber, 1999). Other similar experiments suggest equivocal results mainly because most of them rely on the use of inhibitors. The roles of enzymes in the repair process can also be understood through the mammalian cells, selected for superior resistance to DNA damaging agents. The theory behind this dictates that if topoisomerase play dire roles in repair, then that only mean that, these cell lines that are resistant to DNA damaging agents must have exceeded topoisomerase levels. Nucleoside analogues The study of nucleoside analogues and their roles brings new insight into functional links in the complex network of the damage response to the DNA. The damage of DNA involves three key disorders namely; A-T ( ataxia-telangiectasia) which occurs due to lack of activation of the protein kinase, ATDL (ataxia-telangiectasia-like disease), which occurs as a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Ataxia-telangiectasia-like disease (ATLD) is a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11, Rad50, and Nbs1 proteins are the core contents of the MRN complex, they are all involved in the initial processing of DSBs. Nucleoside analogues is a simple base that consists of nucleobase bound deoxyrebose sugarvia a beta glycosidic linkage. Nucleosides can be added phosphorus into them by adding primary alcohol group (-CH2-OH), producing nucleosides, which are the building blocks of the DNA and the RNA. These nucleosides can be produced in the body and also can be gotten from ingestion and digestion of supplementing nucleic foods in the diet (Crowe, et al. 2006). In the body they are produced in the liver by the de novo synthesis pathways whereby the nucleotides have been broken down by the nucleotides into simpler form of thymidine monophosphate and into nucleosides like thymidine and phosphate which are later broken down (Crowe, et al. 2006). The diagram below represents a Nucleoside analogue reverse transcriptase inhibitor used to treat HIV. Atoms are represented as spheres and are color-coded: carbon (grey), hydrogen (white), nitrogen (blue) and oxygen (red) (Ataian and Krebs, 2006). In terms of health, these nucleoside analogues are very important because of their vital use in antiviral and anti cancer agents (Ataian and Krebs, 2006). This compound of this canonical base is incorporated by the viral polymerase and later activated in the cells by being converted into nucleotides. Since charged nucleotides cannot easily cross the cell membranes they are administered as nucleosides. RNA has a very low stability level hence it is prone to hydrolysis so it is then bound to a stable alternative nucleoside analogue by using different backbone sugar examples of this analogues include LNA, morpholino, and PNA (Bjorksten, Acharya, Ashman & Wetlaufer, 1971). In the process of sequencing, two deoxynucleotides are utilized and since they contain a dideoxyribose which is a non-canon sugar that lacks 3 hydroxyl groups it accepts the phosphate and terminates the chain for it cannot bond with another base, this happens because the DNA cannot differentiate between it and the deoxyribonucleotide. The nucleoside plays a very vital role in the repair of DNA and in the therapeutic nucleoside analogues it adds on their cytotoxic activity against cancer causing cells by disruption of further DNA synthesis and by incorporation into DNA triggering the apoptosis. These therapeutic nucleoside analogues include gemcitabine, fludarabine and Ara –c. however there are identified molecules that subsequently initiate the downstream cellular responses and recognize the incorporated analogues (Branze & Foiani, 2008). The DNA-PK (dependent protein kinase) forms a complex that interacts with the gemtabine which signals the apoptotic pathways it contains DNA.DNA-PK/Ku were again purified in a protein fraction that binds to gemcitabine-containing DNA in preference to normal DNA. Experiments reveals that this two proteins DNA-PK and DNA-PK/Ku associate physically into a complex. When treated with gemcitabine the result becomes an increase of DNA-PK and protein and also an increase in phosphorylation (Branze & Foiani, 2008). DNA double –strand breaks (DSBs) could be caused by genomes which are subject to numerous exogenous or endogenous DNA-damaging agents, this are critical DNA lesions that could result to cell death or a variety of genetic alterations, including , heterozygosity loss, deletions and translocations, chromosome fusions and chromosome loss which enhances genome instability and could trigger carcinogenesis. Involvement of DNA repair (especially MRN) in providing resistance against top inhibitors and nucleoside analogues In DNA repair, the cells develop an efficient mechanism to cope with DNA damages. Basically there are two major DSB repair mechanisms which are the homologous recombination (HR) and the nonhomologous end joining (NHEJ) an element of this repair mechanism involves the MRN complex which consists of the RAD50 and NBN which also is described as (NBS1 and MRE11; this is now used in the DNA replication signaling of the cell cycle checkpoints and DNA repair. A protein complex called MRN is normally found in yeast (nibrin in humans and Xrs2 in yeast) prior to its role in repair by homologous recombination or non homologous end joining it plays a role in the initial processing of double strand DNA (Budman & Chu, 2005). It also binds avidly to double –strand breaks both in vivo and in vitro and could serve to tether broken ends prior to the repair by non homologous end joining or to initiate resection prior to repair by homologous recombination. There are a number of kinases, like ATR (ataxia-telangiectasia and Rad-3-related) ATM (ataxia-telangiectasia mutated) and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage that has occurred therein. In response to DNA damage the MRN complex participates in activating the checkpoint kinase ATM. ATM activation by the MRN complex implicates production of short singled –strand oligonucleotides by Mre11 endonuclease (Budman & Chu, 2005). Telomere maintenance and genome stability involve the MRN complex as well as the repair kinases. The genome instability disorders like ataxia telangiectasia (A-T), A-T_ like disorder (ATLD) and Nijmegen breakage syndrome (NBS) is a dysfunction of particular elements involved in the repair mechanisms. This is the mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Nucleoside analogues prevent viral replication in the infected cells due to a wide range of antiretroviral products. It is commonly used in Acyclovir, though its inclusion in this category is uncertain as it contains only a partial nucleoside structure as the sugar ring is replaced by an open-structure. These agents are very helpful in the treatments of herpes simplex, HIV, hepatitis B virus, hepatitis C virus for they are able to work as antimetabolites once they are phosphorylated by being similar enough to nucleotides to be incorporated into growing DNA strands but they stop viral DNA polymerase and act as chain terminators (Budman & Chu, 2005). They affect the mitochondrial DNA and also they are not specific to the viral DNA hence side effects that include bone marrow suppression. For nucleoside analogue reverse inhibitors, there is a large family and production by reverse transcriptase of DNA is very different from normal human DNA replication hence the possibility to design nucleoside analogues that are preferentially incorporated by the former. It is also worth to note that some nucleoside analogues however can function both at NRTIs and polymerase inhibitors for other viruses for example in the case of hepatitis B (Budman & Chu, 2005). To treat cancer, less selective nucleoside analogues are used as chemotherapy agents. References: Ataian, Y. and Krebs, J.E. (2006). Five repair pathways in one context: chromatin modification during DNA repair. Biochem. Cell Biol. 84, 490-504. Branze, D., & Foiani, M. (2008). Regulation of DNA repair throughout the cell cycle. Nature Reviews Molecular Cell Biology 9, 297–308. Budman, J; Chu, G (2005). "Processing of DNA for nonhomologous end-joining by cell-free extract". The EMBO Journal 24 (4): 849–60. Bjorksten, J; Acharya, PV; Ashman, S; Wetlaufer, DB (1971). "Gerogenic fractions in the tritiated rat.” Journal of the American Geriatrics Society 19 (7): 561–74. Crowe, F. W., et al. (2006). A Clinical, Pathological, and Genetic Study of Multiple Neurofibromatosis. Springfield, Illinois: Charles C. Thomas Press. Kobayashi, K. et. al. (2005). Involvement of mismatch repair in transcription-coupled nucleotide excision repair. Hum. Cell. 18, 103-15. Lodish, H., et al. (2004). Molecular Biology of the Cell, 5th Ed. New York: Freeman. Leibeling, D. et. al. (2006). Nucleotide excision repair and cancer. J. Mol. Histol. 37, 225-238. Orii, K.E. et. al. (2006). Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development. Proc. Natl. Acad. Sci. USA. 103, 10017-10022. Plosky, B. et. al. (2002). Base excision repair and nucleotide excision repair contribute to the removal of N-methylpurines from active genes. DNA Repair (Amst). 1, 683-696. Reed, S.H. (2005). Nucleotide excision repair in chromatin: the shape of things to come. DNA Repair (Amst). 4, 909-918. Sinha, R. P., & Häder, D. P. (2002). UV-induced DNA damage and repair: A review. Photochemical and Photobiological Sciences 1, 225–236. Schonthal, Axel H. (2004). Checkpoint Controls and Cancer. London: Humana Press. Wang, Z (2001). "Translesion synthesis by the UmuC family of DNA polymerases". Mutat. Res. 486 (2): 59–70. Wilson, TE; Lieber, MR (1999). "Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway". The Journal of Biological Chemistry 274 (33): 23599–609. Read More