Reca Protein Analysis

On December 10, 2015, three profound individuals received the Nobel Prize in chemistry for their work on DNA repair systems. Paul Modrich, Thomas Lindahl, and Aziz Sancar studied how the cell repairs and protects the information held in its DNA; specifically, Paul Modrich focused on DNA mismatch repair. Since DNA constantly replicates, damage and incorrect pairings are expected, but enzymes watch over DNA as it replicates and repair any errors that occur. In the mismatch repair system, enzymes find the mismatch in the copy of DNA, cut the incorrect section out, and replace it with the correct sequence. Paul Modrich’s study of the mismatch repair system has provided the medical field with important information regarding cancer growth and the possibility of a cure.

Although this depiction is not a hundred uncovered in bacteria, percent proven through research, it can be said that a cell is more likely to use NHEJ when it has not replicated yet. On the other hand, the cell leans towards homologous recombination after it has replicated. She also entails that yeast is very useful in studies the processes mentioned above. Additionally, she stated that scientists are involved in efforts to study how mutated genes react to breaks in their structure. Equally important, she recounted that double strand breaks can be monitored by inducing a break within cells. Dr. Friedman details that scientists have gone about this by using a specific, regulated protein that recognizes and chews through DNA

Escherichia coli K12 is a well-studied gram-negative bacteria. First isolated from the human gut, is one of the most used in molecular studies, being the knowledge obtained from studies of E. coli possible to apply to other organisms (Burton and Kaguni, 1997). Some enzymes have a crucial role in the transcription control, the topoisomerases enzymes play an important role in the level of DNA supercoiling, an important property of DNA and chromatin (Gilbert and Allan, 2014). The supercoiling level is modified by a reaction that consists in the transient breakage of the DNA phosphodiester bonds and the movement of strands across the transient breaks (Tse-Dinh and Wang, 1986). In prokaryotes, these enzymes can be classified into two major groups, according to the mechanism of action. While the topoisomerase II (DNA gyrase) generates negative supercoils into relaxed DNA and relax positively supercoiled DNA, the topoisomerase I relax negatively supercoiled DNA. It is important to equilibrate the superhelical state of the cellular DNA (Hirose and Matsumoto, 2000).

Genetic recombination can ultimately lead to the production of traits in the new generation that differ from those found paternally or maternally. One of the benefits of synapsis (or chromosomal crossover) is the formation of new

In this experiment, host NM554, a particular strain of E. coli, was used to cultivate human genes (Dolf, 2013). Through the use of cosmids, plasmids that carry the cos gene, DNA fragments were introduced into the E. coli and packaged into phage particles (McClean, 1998). Pst I is a restriction endonuclease, an enzyme that cuts DNA at restriction sites (Restriction endonuclease). The Pst I digest of human DNA in this study produced the DNA fragments that were examined. The dideoxynucleotide chain-reaction procedure, also known as Sanger sequencing, is the process of lengthening DNA using DNA polymerase to add on deoxynucleotides until a dideoxynucleotide is added on randomly (Rogers). Fluorescence in situ hybridization (represented by the acronym

During the initiation step of the chaperone cycle, N-terminal lid segment closes the ATP binding pocket leading to contact formation at the NTD. After this step, NTD dimerize forming a twisted closed molecular clamp structure that is required for the client folding. Finally, ATP hydrolysis reverts closed structure of Hsp90 back to the initial open structure allowing maturation and release of the client protein. More than 20 co-chaperones including cdc37, HOP, p23, CYP40, HSP70, AHA1 and others regulate the Hsp90 chaperone cycle as they modulate client protein recognition, ATPase activity and the biochemical activity of the clients (1,

16. Explain how the recombination of genes contributes to genetic diversity. Recombination of genes usually involves two different types of genes. During genetic recombination, genes produced tend to have traits or DNA different from the traits or DNA neither related to the original genes involved in the recombination.

The most reasonable inference from the observation that defects in DNA repair enzymes contribute to some cancers is that:

Covalently linked L (long) and S (short) components are broken down into unique long (Ul), flanked by ab and b’a’ repeated segments, and unique short (Us), flanked by ac and c’a’ repeated segments. Homologous recombination between terminal repeats results in four linear isomers at equimolar concentrations (see figure 1). All four isomers, including P (prototype), IL (inversion of the L component), IS (inversion of the S component) and ISL (inversion of both the S and the L component), encode 90 unique transcription genes essential for viral replication (3).

In addition, it is also shown that γ-irradiation causes DNA lesions that are not well repaired in HGPS cells4. As the study shows, molecular details about how repair proteins and checkpoints are responsible for DNA repair mechanism are still unknown4. To unravel the complications in deficiency of DNA repair mechanisms and their relationship with H3K9me3, immunofluorescence staining and confocal microscopy were carried out and it was found that γ-H2AX/53BP1 foci were associated with regions enriched with H3K9me34. Moreover, to analyze the hypothesis that H3K9me3 poses a barrier for defective DNA repair, SUV39 was knocked down by siRNA and it was revealed that DNA damage foci decreased significantly which proved the hypothesis tested4. In addition to this hypothesis, it should be also proven if SUV39H1 levels directly contribute to deficiency of DNA repair and checkpoint response proteins in progeria. For this purpose, levels of SUV39H1mRNA in cells with ectopic progerin were analyzed and it was concluded that SUV39H1 levels were significantly increased in mouse embryonic fibroblasts (MEFs) in comparison to the wild type control4. Based on previous hypotheses, it can be implied that since H3K9me3 is mainly catalyzed by SUV39H1 methyltransferase, levels of H3K9me3 must be higher in Zmpste24 - / -

This msDNA is usually small single-stranded cDNA molecule bound covalently to an RNA molecule which can fold into a stable secondary structure. Eventhough still there are no proof to conform retrons as mobile elements, copies of inserted msDNA can be found in bacterial genome (Lampson and rice 1997). In E. coli overexpression of some msDNAs has increased the number of base substitution mutations and frameshift mutations (Maas et al,. 1994). When most cellular mismatch repair proteins bind with mismatches on msDNA molecules it increase the mutagenic level. During matings of E. coli and Salmonella cells when some msDNAs are overexpressed it increase the recombination between donor and recipient DNA sequences, because of the action of mismatch repair usually the interspecific recombination frequency is normally reduced (Maas et al,. 1996). Even though the function of msDNA is still unknown it helps the bacteria to increase their mutations when the mutations are required for their survival. These retrons

Interestingly, studies from our laboratory and others have demonstrated a stereochemical dependence for dSp processing by enzymes in ODNs. The first instance of a dSp diastereomer difference was reported from our laboratory when Klenow Fragment (exo-) was allowed to insert dATP opposite the isomers for which dSp1-containing ODNs were found to be the more favorable substrate for insertion and bypass.32 This study was extended to in vivo experiments by Neeley, et al. to include pol II and pol IV, also showing more favorable bypass kinetics for dSp1.34 As further evidence for in vivo stereochemical dependency in dSp processing, Henderson, et al. utilized the restriction endonuclease and postlabeling assay (REAP) to determine the mutation profiles for dSp1 and dSp2 in Escherichia coli.28 In the sequence context 5’-d(GSpA)-3’, they found a strong stereochemical dependence in the relative amounts of dG→dT and dG→dC transversion mutations for dSp1 and dSp2. In subsequent experiments, Delaney, et al. showed a somewhat different dependence of mutation profiles on the dSp diastereomers in the sequence context 5’-(TSpG)-3’ using the REAP and competitive replication of adduct bypass assays (CRAB).29,30 An additional observation from our laboratory quantified the digestion kinetics of dSp isomers in the dinucleotides 5’-d(Np[Sp])-3’ (N = A, T, G or C) by nuclease P1 that showed dSp1 was the kinetically preferred substrate in all sequence combinations.33 Lastly, in collaboration

In E. coli, UvrABC endonuclease catalyzes excision repair at sites of pyrimidine dimers or AP sites (a-purinic/a-pyrimidinic sites). This enzyme has an ATPase-dependent translocation activity that allows it to “patrol” DNA searching for errors.

After a BP or LR recombination reaction, this cassette is replaced by the gene of interest to generate the entry clone and expression clone, respectively.

Modification of damaged DNA seems to be an understudied subject, there is much to understand on the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by methylation but much of it is affected on a gene when silenced. When epigenetic modification has been implicated with cancer and aging it causes DNA methylation to also have an impact on the double strand of DNA analysis. Modification as such provoke deteriorating changes like aging found in multicellular organisms and DNA damage may magnify biochemical pathways that regulate a cells growth or control DNA replication with DNA repair. In the article “DNA Damage, Homology-Directed Repair, and DNA Methylation” written by Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may induce a DNA double-strand break within a genome as well as when DNA methylation is induced by homologous recombination that it may somewhat mark its reparation through a DNA segment and protect its cells against any unregulated gene expression that may be followed by DNA damage. The experiments used to demonstration how gene conversion can modify methylation pattern of repaired DNA and when that occurs methylation is able to silence the recombined gene. When exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an induced double strand break that can be found at a specific location or DNA sequence in where the