Dr Lab Report

In this lab, we used two different strains of Saccharomyces cerevisiae (yeast) cells. One of them was, Saccharomyces cerevisiae, UV-Sensitive Strain, G948-1C/U, alpha, rad1 rad18 phr1 ura3 mutant in excision repair (Haploid), while the other was a wild type strain of this yeast. We chose to do this, because it is important to compare UV sensitive yeast cells, which have a mutation that yields them unable to repair themselves, to a wild type that is able to repair itself after UV radiation exposure. According to Conconi, “monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Δ) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after

Colorectal cancer is the third most common cancer among men and women in the United States, and mutations in the MUTYH gene significantly increase the risk of developing polyps that may evolve into cancer.1,2 Biallelic mutations in the MUTYH gene can lead to MYH-Associated Polyposis (MAP), which causes the growth of dozens to hundreds of polyps, furthering increasing the risk for colon cancer.2 Meanwhile, recent studies have shown that both biallelic and monoallelic mutations can contribute to bladder, ovarian, gastric, hepatobiliary, endometrial, and breast cancer.3 The MUTYH gene itself codes for the MYH glycosylase enzyme, which repairs mistakes in DNA caused by reactive oxygen species.3,4 Also a sign of oxidative stress, the oxidation product, 8-oxo-7,8-dihydro-2-deoxyguanosine (OG), mimics thymine, eventually matching with adenine and resulting in a complete loss of the cytosine-guanine pair.4 MUTYH removes the undamaged A base from the mismatched pair, aiding in the correction of such damage.4 However, when MUTYH is mutated, there is an increase in G to T mutations, which can eventually affect the tumor suppressor genes APC and K-ras and lead to tumor formation.4 The MUTYH variants Y165C and G382D are the most common mutations seen in individuals with MAP, and for this reason are of great interest in research working towards reducing the risk of colorectal cancer.2,4 In recent years, the CRISPR/Cas system for gene editing has become the preferred method for

Chromatin rearrangement takes place during repair to increase accessibility of damage to repair proteins. Activity of histone acetyltransferases and chromatin remodeling complexes seems to be essential for nucleosome rearrangement during repair. Once repair is completed, reconstitution of nucleosomes is required to recover primary chromatin structure. Such chromatin assembly is coupled to repair DNA synthesis. DNA damages induce some modifications to chromatin structure (e.g. phosphorylation of a histone H2A variant in response to DNA double-strand breaks). Such chromatin modifications may serve as signals recognized by DNA repair

Of these proteins include Breast-Cancer susceptibility gene 1 (BRCA1), Breast-Cancer susceptibility gene 2 (BRCA2), and RAD51 recombinase (RAD51). BRCA1 and BRCA2 mutations are most commonly associated with predispositions to breast and ovarian cancers; these genes are found to play fundamental roles in cellular processes such as DNA repair via homologous recombination (HR)29. Within the repair pathway of DSBs, BRCA1 is phosphorylated by ataxia-telangiectasia mutated kinase (ATM) in response to DNA damage30, 31. BRCA1 then relocates to the site of damage and recruits BRCA2. BRCA2 is a very large protein, weighing 384kDa, which directly co-localizes with RAD51 and acts as a scaffold on top of the DSB to allow RAD51 to stabilize and directly repair the DSB through HR (Figure 1). RAD51 protein possesses many biochemical activities required for HR including the ability to promote joint molecule formation and DNA strand exchange between homologous DNA molecules32. Many studies have reported that a lack of BRCA activity can lead to irregularities in DNA repair including radiosensitivity, defects in recombinational repair, and obstruction in the localization of repair proteins like BRCA2 and RAD5124, 33, 34. Recently, one study has shown that BRCA1 levels are reduced in the brains of Alzheimer’s disease patients and that this depletion led to increased neuronal DSBs in mice35. It was also

In response to γ-irradiation mediated DNA damage, ATM kinase activates the tumor suppressor protein, p53, via phosphorylation. Once activated, p53 initiates two negative feedback loops. The first negative feedback loop is caused by p53 activating the expression of the E3 ubiquitin ligase Mdm2, allowing Mdm2 to target p53 for degradation. The second negative feedback loop is the result of p53 activating the expression of the phosphatase Wip1, which dephosphorylates p53 and therefore reduces its stability. Wip1 also targets ATM kinase, which requires phosphorylation in order to be in the active state. Therefore, the increased activity of Wip1 silences ATM kinase, which prevents the activation of p53. If DNA damage persists in the cell, ATM kinase activates p53 once more after Wip1 expression levels have subsided. Pulsing of p53 activity results in cell-cycle arrest and DNA repair, and continues until DNA damage has been completely repaired.

The single-stranded DNA-binding proteins (SSB) play an important role in DNA replication, repair and recombination, they are found in D. radiodurans as homodimers

Nucleotide excision repair genes are related to Xeroderma Pigmentosum. These are the genes that repair DNA damage caused by mutagenic agents such as UV. Previous studies have shown that mutations to these specific set of genes are highly related to clinical symptoms in XP patients. These genes are: DDB2, ERCC2, ERCC3, ERCC4, ERCC5, POLH, XPA and XPC.

Cancer, also known as malignant neoplasm, the hallmark characteristic involves uncontrolled proliferation of cells. Under normal conditions cell grow, divide and die, but in cancer cells the defect takes place at the gene level leading to the formation of an abnormal DNA. As genes are the basic control machines of the cell, alteration of any kind may bring devastating consequences, or malignancy. Moreover, in normal cells damage of any kind is taken care by the repair system but in cancer cells, the repair function is altered, hence damaged DNA is not repaired, occurs, cells divide continuously without displaying senescence, generating a series of abnormal cells (Alberts, 2007).

Saccharomyces cerevisiaes, or baker’s yeasts, unicellular fungi are useful in understanding genetics and molecular biology, due to the ability to quickly map a phenotype-producing gene to a region in their genome. Yeast mutants are used a tool for the study of cellular function, DNA repair mechanisms and cell cycle control. As a model organism, S. cerevisiae is one of the simplest eukaryote organism, having not only most major signaling pathways conserved, but also consisting of a genome of approximately 12.1 million base pairs in sixteen chromosomes. S. cerevisiaes, like other model organisms, have properties that make them suitable for biological studies: rapid growth, easy mutant isolation, a sequenced genetic system and a versatile DNA transformation system, as homologous recombination is used almost exclusively as their DNA repair mechanism.

DJ-1 is implicated in many cellular processes, has a cytoprotective function and acts as a redox sensor. Cysteine 106 within DJ-1 is a conserved and the most reactive residue and is susceptible to oxidation induced by ROS. Mutations within DJ-1 such as methionine to isoleucine substitution at position 26 (M26I), a glutamic acid to aspartic acid (E64D) substitution, substitution of proline for leucine (L166P) or DJ-1 deletions are involved in disease pathophysiology (24, 25). While DJ-1 cytoplasmic and nuclear functions have been extensively studied, its role in mitochondria remains poorly characterized. Therefore, in this SA, we will determine the function of DJ-1 in mtDNA damage repair.

Double-strand breaks in DNA (DSB) constitute one of the greatest threats to cell viability. In eukaryotes DSBs are mainly repaired thanks to the process called "Non-homologous end-joining" (NHEJ), and consists of the direct ligation of the generated ends after the break without the need for a “template”. The objective of this paper is to discuss the different mechanisms available to correct DSBs in DNA. The components that participate in these repair mechanisms play a fundamental role in maintaining genomic stability (Specially in NHEJ). Furthermore, these mechanisms participate in programmed DNA breaks that mediates biological processes such as V(D)J recombination. During NHEJ the Ku protein binds to the generated ends and lays

3A). However, other nucleotides like deoxycytidine (Fig. 3A) are glycated at high α-oxoaldehyde concentrations forming 5-glycolylcytidine (Frischmann, M. et al 2005; Kasai, H. et al 1998; Krymkiewicz, N. 1993). Inter-strand cross-links in duplex DNA (Seidel W and Pischetsrieder M 1998; Kasai, H. et al 1998) and DNA–protein cross-links in human keratinocytes were reported at higher MG concentrations. A recent in vitro study has demonstrated that CEdG (Fig. 3B) formation in DNA can hinder the Klenow fragment-mediated DNA synthesis (Cao, H. et al 2007). Due to AGEs the hydrogen-bonding property of the modified guanine is affected, resulting efficient incorporation of purine than pyrimidine nucleotides (Cao, H. et al 2007). Addition of bulky AGEs groups on the N2 position of guanine altered the Watson–Crick base pairing characteristic of these nucleobases resulting in destabilization of duplex DNA. The presence of dGTP-AGEs in DNA may change DNA structure, leading to the formation of abasic sites and DNA strand breaks (Pischetsrieder, M. et al 1999; Seidel W and Pischetsrieder M 1998; Cao, H. et al 2007). DNA glycation generates several nucleotide adducts that increases oxidative stress by the generation of hydrogen peroxide, superoxide, and hydroxyl radicals (Mistry, M. et al 2003; Thornalley, P.J. 1996). Recently a study demonstrated an accumulation of

Oncogenes, tumour suppressor genes and DNA repair genes are three types of cancer genes. People may inherit a mutated version of one of these genes which will make them more likely to develop a certain type of cancer. Oncogenes cause cells to grow out of control and promote cancer cell growth. They are a damaged version of the normal proto-oncogenes in our bodies. Tumour suppressor genes are genes that would, in a healthy person, protect us from cancer. They help us stop cell growth and control cell death. When these cells are missing or damaged they cause cell growth, division and death to be uncontrolled. According to the Canadian Cancer Society, “nearly 50% of all cancers are thought to involve a damaged or missing tumour suppressor gene.” DNA repair genes are responsible for repairing damaged genes. During copying of DNA, if mutations occur, they are there to fix the mistakes. These three genes are the main causes of cancer related mutations.

Double stranded breaks (DSB) occur frequently in eukaryotic cells. It can be caused by many factors such as ultra violet radiation, reactive oxygen species, ionizing radiation et cetera (Lieber,2010). DNA damage leads to rapid growth of tumor leading to cancer. Hence it is very important to repair it before the cell undergoes further division. Two mechanisms of repair system can occur in the cell – homologous recombination (HR) and non homologous DNA end joining (NHEJ). These repair systems along with their mechanism and the repair factors associated with it has been analyzed in this paper. In order for repair factors to access the DNA that are packaged, chromatin remodelers are essential to open the DNA. One way of DNA being

While DNA is being repaired there is a mutation that interferes, causing this inconvenience; so