MRN-Dependent and Independent Topoisomerase Removal Mechanisms - Term Paper Example

Student Name: Instructor’s Name: Title: Analysis of MRN-dependent and -independent topoisomerase removal mechanisms: implications for resistance against camptothecin and etoposide derivatives Course: Institution: Analysis of MRN-dependent and -independent topoisomerase removal mechanisms: implications for resistance against camptothecin and etoposide derivatives Literature Review Nucleoside analogues These refer to a variety of antiviral products that are utilized in preventing viral replication within the cells that are infected. An example of a nucleoside analogue is Acyclovir, even though its inclusion within this classification is uncertain since it has just a partial nucleoside structure since an open-chain structure replaces the sugar ring (SEER Training Modules 2012). Nucleoside analogues are utilized in treatment of hepatitis B virus, HIV as well as hepatitis C virus. After their phosphorylation, they function as antimetabolites through being equivalent to nucleotides to be integrated into developing DNA strands. However, they function as chain terminators and hence they discontinue viral DNA polymerase. However, the nucleoside analogues have sides effects including suppressing the bone marrow since there are not specific to viral DNA in addition to affecting mitochondrial DNA. Basically, nucleoside analogue reverse transcriptase inhibitors (NRTIs) consists of a big family since production of DNA through reverse transcriptase varies greatly from usual human DNA replication and thus designing of nucleoside analogues that are preferentially integrated through the former is possible. Nonetheless, some nucleoside analogues are able to function as NRTIs as well as polymerase inhibitors for other viruses such as hepatitis B. On the other hand, nucleoside analogues that are less selective are utilized as chemotherapy agents during cancer treatment. Resistance to nucleoside analogues can develop fast with even one mutation. Mutations take place within the enzymes that lead to phosphorylation of the drug for its activation (SEER Training Modules 2012). Cancer Cell Basically, cancer occurs when there is uncontrollable growth of the cells. There are several such uncontrolled cells’ growth and the cancer classification is done according to the cell type the cancerous cell initially attacked. Cancer interrupts the normal growth of the cells as well as the functioning of the cells since tumors develop and destroy other cells. In the process of destructing the body functions, the cancerous cells grow forming massive cells that develop interrupting other functions of the body such as nervous and circulatory system through production of enzymes or hormones that are detrimental to the usual body function. Cancerous cell can remain and go on increasing at the primary cell and not invade other body cells and these are benign or can move and attack other cells and in this case they are referred to as malignant. The malignant ones are the dangerous ones since they can migrate, attacking healthy cells through metastasis (Krogh & Symington 2004). Factors contributing to cells becoming cancerous include, mutations, exposure to carcinogens, some viruses in addition to inheritance. These factors impact the normal growth of the cells, cell divisions as well as death process and hence confer abnormal constant cell growth. Cancer is one of the leading causes of mortality yearly and interferes with the economy and therefore more funds are supposed to be assigned to cancer research to be able to establish cheaper diagnostic methods and treatment emphasizing on cancer prevention. Within a cancerous cell, mutation of numerous genes occurs resulting to a defective cell. For example, dominant mutation results from an abnormality within a single gene within a pair (Watson 2008). In such a case, the mutated gene generates a protein that is defective and this results into the growth receptor on the surface of a cell turning on, when actually there is no growth factor whatsoever. In p53 gene, a normal protein is produced and it stops division of the cells and hence it controls the growth of the cells. The key role of p53 gene is repairing the defective cells in order to prevent the likelihood of cancerous cells growing. The gene which is normally referred to as anti-oncogene can remain on tissue cell neighboring the tumor cells or can circulate within the blood, attacking other cells that are not close to the primary cancerous cell where its division goes on, and this process is referred to as “metastasize”. In contrast, when insignificant defects take place within the DNA, an abnormal DNA is formed and hence flawed proteins are produced (Krogh & Symington 2004). Since cancer has recently turned out to be a common disease, education of people regarding the disease is needed and also information about cancer should be availed to everybody so that the information is readily available. In such an instance, it will be easy in preventing the disease and the people suspecting to have the disease are supposed to get a medical examination and such individuals are supposed to be encouraged to be examined medically regularly. In order to make this a reality, the government ought to invest within suitable and latest cancer facilities to enable everybody access in particular the poor people. Replication Figure 1: DNA replication During DNA replication, the double helix unwounds and every strand acts as a template for the next strand and bases are then matched for synthesizing new strands. The replication process begins when a double-stranded DNA molecule produces two matching copies of the DNA molucule. Replication process states at a location known as origins and the DNA unwinding ar the origin and synthesising on new strands produces a replication fork (Doublie 1990). Figure 2: Replication fork DNA polymerase synthesizes the new DNA through additional of nucleotides equivalent to the template strands and also there are some proteins that are allied to the replication fork and help in initiating and continuing the synthesizing of DNA. Replication of DNA can be carried out in vitro where DNA polymerases, obtained from cells along with artificial DNA primers are utilized in initiating synthesizing of DNA at known sequences within a template molecule. The polymerase chain reaction is a laboratory technique that uses this artificial synthesis within a cyclic means in amplifying a particular target DNA fragment from a DNA pool (Lambart, Froget & Carr 2007). DNA repair Figure 3: DNA damage caused by multiple broken chromosomes DNA repair refers to procedures whereby a cell identifies and rectifies damage to the DNA molecule encoding its genome. In human cell, usual metabolic activities as well as environmental aspects like radiation can lead to DNA damage which may lead to mutations. Accordingly, the process of DNA repair is always active since it responds to damage within the structure of DNA. In case the usual DNA process fails, and if cell death does not take place, irreparable DNA damage might take place and this includes double-strand breaks as well as DNA cross-linkages. DNA repair rate depends on several factors and the factors consist of type of the cell, age of the cell in addition to the extracellular environment. A cell that has built up a huge quantity of DNA damage or one that does not repair the damage its DNA incurs efficiently, can go into the one of following likely states: Irreversible dormancy state Cell suicide Uncontrolled cell division, which can result into a cancerous tumor The ability of a cell to carry out DNA repair is important to the genome’s integrity and hence it is important to the normal functioning of the organism. Several studies have shown that genes that formally indicated to affect life span have turned out to playing a role in the DNA damage repair as well as protection. If the molecular lesions within the cells forming gametes are not corrected can lead to mutations into the offspring’s genomes and hence affect the evolution rate. DNA repair mechanisms Figure 4: Single-strand DNA damage Figure 5: Double-strand DNA damage Depending on the form of the damage that occurs on the double helical structure of the DNA, different types of repair mechanisms can be used in restoring the lost information. In case it is possible, cells utilize the unmodified complementary DNA strand or the sister chromatid as a template for recovering the primary information. If the template cannot be accessed, cell use an error-prone mechanism referred to as translesion. DNA damage tampers with the spatial pattern of the DNA double helix and it is possible for the cell to detect such interferences. After the localization of the damage, specific DNA repair molecules attach to the damage location and induce other molecules to attach and finally forms a complex that facilitates repairing of the damage (Nakagawa 2002). Direct reversal Cells can eliminate the DNA damage through chemical reversal. Direct reversal mechanisms are specific to the kind of the damage and there is phosphodiester backbone breakage. For example, when pyrimidine dimers are formed after UV light irradiation leading to an anomalous covalent bond between adjoining pyrimidine bases, the photoreactivation process is able to reverse such kind of damage directly by using enzyme photolyase, which is activated by the UV light (300-500 nm wavelength) and hence instigates catalysis (Nakagawa 2002). Single-strand damage Figure 6: Base-excision repair enzyme uracil-DNA glycosylase. The yellow one is the uracil residue In this form of repair mechanism, when just one strand is damaged, the other strand is utilized as a template for direction correction of the damaged DNA strand. For the repair to take place, there are several excision repair mechanism that delete the damaged nucleotide and replacing it with an undamaged nucleotide. These mechanisms include: Base excision repair: this mechanism repairs any damage on a single base resulting from oxidation, alkylation, hydrolysis and deamination. The base that has the damage is eliminated through a DNA glycosylase and then the missing tooth is identified by enzyme AP endonuclease and this enzyme cuts the phosphodiester bond and the resythesis of the missing part is done by a DNA polymerase while DNA ligase seals the nick. Nucleotide excision repair: in this case, the bulky lesions that distort the helix, like pyrimidine dimers are recognized. A specialized type of Nucleotide excision repair deploys the Nucleotide excision repair enzymes to genes being undergoing transcription. Mismatch repair: this rectifies the errors of DNA replication as well as DNA recombination that lead to mis-paired nucleotides (Nakagawa 2002). Double-strand breaks Double-strand breaks, whereby the two strands are severed are extremely dangerous to the cell since this can cause rearrangement of the genome. Mechanisms that repair the double-strand breaks include non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) in addition to homologous recombination (Nakagawa 2002). Figure 7: DNA ligase repairing chromosomal damage DNA ligase binds the broken nucleotides through catalyzing an inter-nucleotide ester bond to be formed between the phosphate backbone and the deoxyribose nucleotides. In NHEJ, DNA Ligase IV leads to the formation of a complex along with cofactor XRCC4 and joins the two ends directly. For a precise repair, NHEJ depends on microhomologies that are the sing-stranded DNA ends for joining to take place. When the overhangs are compatible, repair is normally precise. NHEJ is some cases introduces mutations when repair is taking place and if the damaged nucleotides are lost at the break location deletions can occur and hence non-matching termini can be joined forming translocations. In particular, NHEJ is essential prior to DNA replication because of the absence of a template for repair through homologous recombination. Topoisomerases introduce the single as well as the double-strand breaks during the changing of the DNA’s super-coiling state and this is mostly common within areas adjacent to an open replication forks. These kinds of are not regarded as DNA damage since they are a natural intermediate within the topoisomerase biochemical mechanism and are instantly repaired through using the enzymes that created them (Nakagawa 2002). Translesion synthesis This is a DNA damage tolerance mechanism that enables the DNA replication machinery to replicate past DNA lesions like thymine dimers. This mechanism entails alteration of the normal DNA polymerase for specialized translesion polymerases regularly with huger active locations that enable bases to be inserted opposite the nucleotides that have been damaged. Post-translation modification of the replication processivity factor PCNA mediates the polymerase switching. Normally, translesion synthesis polymerases have higher probability of inserting the incorrect bases on the templates that are not damaged when compared to regular polymerases (Sinha & Hader 2002). DNA damage checkpoints Normally, activation of cell cycle checkpoints occurs following DNA damage. This process stops the cell cycle for some time and hence offers the cell time to repair the damage before going on with division. DNA damage checkpoints take place at the G1/S as well as G2/M locations. There is also an intra-S checkpoint. Kinases ATM and ATR control the checkpoint activation whereby ATM establishes a response to DNA double-strand breaks as well as disruptions within chromatin structure whereas ATR basically establishes a response to the replication forks that are stalled. . Kinases ATM and ATR phosphorylate downstream targets within a signal transduction cascade, finally resulting to cell cycle arrest. A category of checkpoint mediator proteins consisting of BRCAI, MDCI in addition 53BPI has been discovered. The proteins are needed transmission of the checkpoint activation signal to downstream proteins. P53 is a vital downstream mark of ATM along with ATR. P53-dependent and p53-independent mechanisms induce the cyclin-dependent kinase inhibitor p21 and it is bale to arrest the cell cycle at the G1/S as well as G2/M checkpoints through deactivating the cyclin/cyclin-dependent kinase complexes (Lambart, Froget & Carr 2007). Pathological effects of poor DNA repair Figure 8: DNA repair rate as a cell pathology determinant Experimental studies using animals having DNA repair defects have indicated reduced life span as well as raised incidence of cancer. For instance, a rat having a default within the dominant NHEJ pathway in addition to telomere maintenance mechanisms often acquire lymphoma as well as infections and as a result have shortened life spans. Likewise, a rat having a default within a major repair and transcription protein responsible for the unwinding of DNA helical have demonstrated premature inception of illnesses associated within aging and as a result have a shorter life span. nonehtless, not all DNA repair defaults lead to precisely predicted effect. In case the rate of DNA damage is higher than the cell’s ability to repair the damage, errors built up and this overwhelms the cell resulting to an early senescence, cell death or cancer (Petrakis 1999). Rate of evolutionary change In some instance, DNA damage is not repaired and if repaired it’s through an error-prone mechanism that leads to a change form the primary sequence. When this takes place, mutations can propagate into the genomes of the progeny of the cell. In case such an incident takes place within a germ line cell that will finally produce a gamete, the mutation can be passed to the offspring. Evolution rate in a specific gene is a function of the rate mutation and hence both the rate and precision of DNA repair mechanisms have an effect on the evolutionary change process (SEER Training Modules 2012). DNA repair and cancer There are about thirty four Inherited human DNA gene mutations that elevate risk to cancer. Specifically, Hereditary nonpolyposis colorectal cancer is strongly allied to mutations within the DNA mismatch repair pathway. In addition, BRCA1 and BRCA2 are mutations that confer a largely elevated risk to break cancer on carriers and they are allied to many DNA repair pathways, in particular NHEJ as well as homologous recombination (Takashima et.al 2002). Cancer treatment processes like chemotherapy and radiotherapy function through overwhelming the ability of the cell to repair DNA damage and this leads to cell death. Cells that in most cases divide constantly and fast, a typical aspect in cancer cells are preferentially affected. However, these treatments have side effects where other cells that are not cancerous but divide rapidly, for example stem cells within the bone marrow are affected too. Modern cancer treatments try localizing the DNA damage to only the cells and tissues that are linked with cancer, through physical ways, for example by concentrating the therapeutic agent within the tumor area or through biochemical ways, through exploitation of a feature that is specific to cancer cells within the body. Longevity and caloric restriction Figure: Most life span influencing genes affect the rate of DNA damage Several specific genes have been identified as having an influence on variations within life span in a population of organisms. The impact of these genes mostly depends on the environment, particularly on the diet of the organism. Caloric restriction reproducibly leads to a longer lifespan within different organisms, probably through nutrient sensing pathways as well as reduced metabolic rate. The molecular mechanisms through which such restriction lead to longer lifespan is not clear yet but the behavior of several genes known to take part in DNA repair is altered during caloric restriction. For instance, the mammalian homolog of SIR-2 induces downstream DNA repair factors taking part in NHEJ, which is an activity that is particularly promoted during caloric restriction. A number of studies have shown that caloric restriction has been associated with the rate of base deletion repair within the nuclear DNA, even through comparable effects haven’t been observed within mitochondrial DNA. C. elegans gene AGE-1, which is an upstream effector of DNA repair pathways, bestows noticeably expanded life span during free-feeding state but results to a reduction in reproductive fitness during caloric restriction (Petrakis 1999). Topoisomerases Topoisomerase type I and type II are the enzymes responsible for the regulation of DNA over-winding or under-winding. Problem in the winding of DNA occur because of the intertwined nature of its double helix. For instance, when replication of the DNA is taking place, DNA gets over-wound in front of a replication fork. In case it is not bated, the tension would ultimately break up replication to a stop. Topoisomerases helps in overcoming these kinds of topological problems resulting from the double helix through binding to either single-stranded or double-stranded DNA and cutting DNA’s phosphate backbone. As a result, the intermediate break enables untangling or unwinding of the DNA and at the end of these procedures, the backbone of the DNA is released again. Because in general the chemical composition as well as the connectivity of the DNA remains the same, both tangled and untangled DNAs are chemical isomers and are only different within their global topology. Basically, topoisomerases are isomerase enzymes acting on the topology of DNA. Function The DNA has a double-helical configuration that makes it hard for the separation of the DNA strands and yet the strands must be separated through helicase proteins for the other enzymes to be able to transcribe the sequences responsible for encoding the proteins or for the replication of the chromosomes. The circular DNA where the double helix DNA is bound the two strands are topologically connected. Otherwise matching DNA loops, with varying number of twists are topoisomers and cannot be inter-converted through any procedure that doesn’t entail separation of DNA strands. Topoisomerases catalyze and guide the DNA unwinding through creation of transient breaks within the DNA utilizing a conserved Tyrosine as the catalyst. Inserting viral DNA into chromosomes as well as other types of recombination can also necessitate topoisomerases action. Clinical significance of topoisomerases Several drugs function by interfering with the topoisomerases. Some drugs used in treating cancer known as topoisomerase inhibitors function through interference of mammalian-form eukaryotic topoisomerases within cancerous cells. This stimulates breakage of DNA that eventually results to death of the cells. The effect of damaging the DNA, outside of its potentially curative aspects, might result into secondary neoplasms within the patients (SEER Training Modules 2012). Topoisomerase I Topoisomerase type I is responsible for cutting the DNA double helix and then relaxation takes place and this leads to re-annealing of the cut DNA strand. When the DNA strand is cut, a part of the molecule on one side of the cut DNA rotates around the strand that is not cut and hence reduces stress from a lot of too little twist within the helix. This stress occurs when the DNA strand is “super-coiled” or uncoiled to or from higher coiling orders. Figure 9 Structure of the Topo I/DNA complex. PDB ID = 1A36 Topoisomerase I are classified into two subclasses: Type IA topoisomerases and type IB topoisomerases, which use a controlled rotating system. Type IA topoisomerases normally form a covalent intermediate with the DNA’s 5’end whereas type IB topoisomerases form a covalent intermediate with the DNA’s 3’end (Hsian et.al 1985). Type II topoisomerases Type II topoisomerases is responsible for cutting the two strands of one DNA double helix and then passes another unbroken DNA helix through it and then re-anneals the cut DNA strand. Type II topoisomerases is also divided into two sub-categories namely, type IIA and type IIB topoisomerases and they both have equivalent structure as well as mechanisms. Normally, type II topoisomerases use ATP hydrolysis (Hsian et.al 1985). Topoisomerase IA IB IIA IIB Metal Dependence Yes No Yes Yes ATP Dependence No No Yes Yes Single- or Double-Stranded cleavage? SS SS DS DS Cleavage Polarity 5' 3' 5' 5' Change in L ±1 ±N ±2 ±2 Figure 10: The structure of supercoils a) Positive supercoils b) Negative supercoils  c) The positive supercoil within bacteria in DNA replication. Type I and type II topoisomerases usually change the connecting number (L) of DNA. Type IA topoisomerases alter the connecting number through one by one, type IB as well as type IC topoisomerase alter the connecting number through any inter, whereas type IIA and type IIB topoisomerases alter the connecting number by two (Malik & Nitiss 2004). Topoisomerase inhibitors Topoisomerase inhibitors refer to therapeutic agents that are devised to tamper with the functioning of topoisomerase enzymes. Recently, topoisomerases have turned out to be popular targets during cancer chemotherapy treatment. The topoisomerase inhibitors hinder the ligation process during cell cycle and this generates single and double stranded breaks and hence damaging the genome’s integrity. When these breaks occur, apoptosis occurs followed by eventual cell death (Hartsuiker, Neale & Carr 2009). MRN Complex From the time MRN complex was discovered, numerous researches have been done to find out if it is used in controlling cancerous cells just like it takes part in repairing DNA defects. This is critical more so in the virus-induced cancerous cells. Within the normal cell cycle, adenosine tri-monophosphate (ATM) plays a role in repairing DNA damages. The activated ATM repairs the damages on DNA molecules. In any case if the ATM is deactivated, damaged DNA pieces build up and this lead to tumors developing. MRN is needed or takes part in activating ATM, even though the mechanism has not been understood entirely at present but there are some hints that MRN complex can be utilized efficiently in controlling DNA damages and thus it can prevent tumors from growing. In order for the MRN to function effectively, the hssB1 molecule should be there since the molecule takes part in binding the MRN complex to the double strand break through attaching to the NBSI part of the MRN complex and therefore the activities of endonuclease are stimulated (Akamatsu et.al 2008). The “Mre11/Rad50/Nbs1, (MRN), which is a protein complex participates in numerous early responses to DNA damage. The protein spo11 is responsible for initiation of “Saccharomyces cerevisiae,” which is a meiotic recombination that results into a DSB within the DNA. Spo11 stays covalently attached to the 5’ ends of the break and the removal is through cleaving endonucleolytic in order to instigate successive DSB end resection as well as meiotic recombination within the Saccharomyces cerevisiae rad50s point mutant (Limbo et.al 2007). This separates function mutant having serious damages within meiosis but just minor damages within mitotic DNA repair, meiotic DSBs created, but this mutant does not have the ability of removing Spo11 from meiotic DSB ends. And because “a nuclease non functional mre11-D56N mutant” has defects in regard to meiotic DSBs reviving, it has been hinted that the nuclease activity of Mre11 plays an important role in removing Spo11 even though this has not been validated through experiments. Even if there is compatibility in connection to the MRN complex taking part in removing Rec12Spo11, experimental demonstration is necessary (Hartsuiker at.al, 2009). Several research studies have shown that if the functions of topoisomerases are inhibited, the normal DNA replication and transcription procedure would be altered. There are suggestions that the tumor cells divide incessantly due to the fact that there is disruption of its check and balance. Having this information, there have been studies on the impact on inhibition of topoisomerase enzymes with the aim of preventing the cancerous cells from growing. The study outcomes have indicated that drugs inhibiting the usual functioning of these enzymes indeed impede replication as well as transcription of the cells and this leads to death of the cells and therefore spreading and growing of the tumor cells is controlled (Liu, Pouliot & Nash 2002). The topoisomerase enzymes play an important role in the DNA metabolism, where these enzymes modify the number of supercoils within DNA, which is an important prerequisite for the cell processes in transcription in addition to replication. Generally, there are significant structural imminent coming from the crystal structure of topoisomerase l, whereas modeling links are starting to establish a probable configuration for the action of topoisomerase II. Normally, there are 5 topoisomerase enzymes that are deemed to be efficient antitumor targets of the drugs. For instance, when the level of mean Top2_ was analyses in patients, the indications were that there were peak elevations between 2-6 days after the therapy process was commenced. Furthermore, a previous study established that plasma camptothrecin concentrations may be playing an important role within the alterations of the protein levels of PBMNC Top 1. In this study, topoisomerase proteins levels were examined within patient PBMNCs as well as the examination of the pharmacokinetics of irinotecan in addition to its metabolites. Cleaving and resealing of DNA’s phosphodiester backbone was done and covalent enzyme-DNA linkage was formed and this enabled another single or double strand to be passed using the nicked DNA. In addition, the study established that Topo l attached to single strand breaks as well as Topo I–irinotecan–DNA cleavable complex did not have fatal effects on the cells by itself (Lambart, Froget & Carr 2007). Camptothecin (CPT) This is a cytotoxic quinoline alkaloid that works by inhibiting the topoisomerase I. this therapeutic agent illustrates key anticancer activity but it has low solubility as well as adverse drug reaction. The Camptothecin analogues utilized in cancer treatment include topotecan and irinotecan. Camptothecin binds to the topoisomerase I and DNA complex and this results to a ternary complex and hence stabilizes it. This hinders DNA re-ligation and hence leads to damage of DNA and this causes apoptosis. Camptothecin bind the enzyme and DNA with hydrogen bonds. The E-ring of the Camptothecin structure interacts from three varying locations with the enzyme. The hydroxyl group within position 20 outlines hydrogen bond to the side chain on aspartic acid number 533 within the enzyme. It is important that the arrangement of the chiral carbon is (S) since (R) is inactive. The lactone bonds with two hydrogen bonds to the amino groups on arginine 364. The D-ring makes interactions with the +1 cytosine on non-cleaved strand and hence stabilization of the topo 1-DNA covalent complex is achieved through hydrogen formation (Hartsuiker, Neale & Carr 2009). The hydrogen bond forms between carbonyl group within location 17 on the D-ring as well as amino group on the pyrimidine ring of the +1 cytosine. When the single-strand breaks is converted to double-strand breaks, toxicity of Camptothecin take place during the S-phase when the replication fork runs into the cleavage complexes that are form from the DNA and Camptothecin. Etoposide This is an anticancer therapy that inhibits topoisomerase. Etoposide makes use of the usual mechanism action of topoisomerase II, which assists in unwinding DNA and hence leads to the breakage of the DNA strands. Normally, cancerous cells depend on topoisomerase II more as compared to the cells that are healthy. Etoposide is used in chemotherapy treatment of different forms of cancer, for instance lung cancer, lymphoma, Ewing’s sarcoma and such. Etoposide is regularly administered with other drugs and at times it is utilized within a conditioning regiment before a blood stem cell transplant. Etoposide acts by forming a ternary complex with DNA as well as the topoisomerase II and thereby hindering re-ligation of the DNA strands. As a result, this leads to error within synthesis of the DNA and hence induces cell death of the cancerous cell (Malik & Nitiss 2004). 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M, and Armstrong j, 2008. ‘Gene tagging and gene replacement using recombinase-madiated cassette exchange in schizosaccharomyces pombe.’ Gene journal, vol 407 p 63-74. Read More