Ap Endonuclease Research Paper

Insertion- this is the addition of an extra base onto an existing DNA strand. The resulting m-RNA base and the subsequent amino acid alignment are altered and no polypeptide chain is formed since none of the intended amino acid have been linked, the impact will have on protein called Frameshift.

The enzyme UvrABC endonuclease, comprised of three polypeptides encoded by the three genes uvrA, uvrB and uvrC, cuts the damaged DNA producing an oligonucleotide of 12-13-mer.

Moreover, more exams and testing was conducted with results that showed how mitochondrial DNA polymerase had all the Y955C mutation in common. This mutation was only found in people who had or showed signs and symptoms of PEO (NIH, 2011). Throughout DNA replication in mitochondria, the strands in DNA are regularly repaired by the formula found in DNA polymerase called exonuclease. Exonuclease works to remove dNTP that was placed incorrectly and was replaced with the correct ones. The mutator Y955C pol gamma as the DNA polymerase, represents a transgenic over-expression of the pol gamma exonuclease-deficient. This process makes it impossible to be able to correct the pairing of dNTP (Ponamarev M. V.,

The gRNA directs the Cas9 endonuclease to the specific sites in the genome and causes a double-strand break (DSB). The host cells repair the DNA damage through the non-homologous end joining pathway and can introduce insertions and deletions in the target site in the process (Yu et al., 2017).

Ligase is essential for putting together okazaki fragments during replication, and also for completing short- patched DNA synthesis occurring in DNA repair process.

In the intrinsic apoptotic signaling pathway, mitochondrial pathway, DNA damage, metabolic stress or the presence of unfold proteins can all be stimuli. These stimuli can pass a signal to the mitochondria system, and finally lead to the formation of the apoptosome. The latter can sequentially activate caspase-9 to cleave the key apoptotic factors caspase-3 and

Inhibits the synthesis of new DNA strands so that no cell replication is possible which normally occurs at ‘S’ phase.

An ubiquitin can change the way a cell functions or even where the call is located. The researchers then use a cdc9 with a wild-type of DNA ligase 1 to determine whether PCNA ubiquitination happens because of the nicked DNA or because of the lack of PCNA-DNA ligase interaction. While reviewing this, they noticed that the cdc9 mutants come to a halt during the later phases of the cell cycle and they collect unligated okazaki fragments. THis proves that PCNA ubiquitination occurs because of the nicked DNA. The nicks are left behind for repairin the last phase of the cell cycle, G2. Although they discovered this, they alos discovered that breaks in the DNA were present in the cdc9 mutants. They caused the replication forks to be held up. These breaks need to be repair via HR. To repair the breaks they suggest RAD52 to be used. Unfortunately, RAD52 was unsuccessful and did not interact with the mutant. They then test if RAD59 can be used to repair the breaks. THey recognize that RAD59 played an important role in supporting the kinase needed for checkpoint activation of the cell cycle, known s Mec1. However, RAD59instead deactivated Mec1 by restraining a protein coding gene called

These breaks are then repaired by the cell in one of two manners: either utilizing non-homologous end joining or homologous recombination directed repair (Figure 2; Hsu et al., 2014). Non-homologous end joining will cause the two ends of the break to join together without the presence of the cleaved portion of DNA between them, resulting in a frame shift mutation (Hsu et al., 2014). A frame shift mutation can be equated to reading a paragraph in a book with a sentence or two missing. The appropriate proteins will no longer be synthesized for that gene, resulting in inactivation, or what is referred to as ‘knockout’ of a gene (Hsu et al., 2014). The second repair mechanism utilizes homologous recombination, during which the section that was broken can exchange DNA with a homologous section within the cell to replace the cleaved sequence (Hsu et al., 2014). This is helpful in genomic manipulation and potential repair of mutations, that otherwise would have led to

DNA, the double polymer composed of nucleotides, is directly responsible for the production of proteins via RNA. When a change occurs in a stand of DNA the resulting strand is mutated. An example of this could be a in the form of a frameshift mutation where a nucleotide sequence is either inserted or deleted from a sequence resulting in a new, and likely defective stand. This defective strand would then transcribe defective RNA, which would lead to mutated proteins being produced. These mutated proteins could have devastating effect on the body allowing cells to go unchecked with the end product being tumors or

Gene knockdown is the technique where the expression of one or more of an organisms genes are reduced by the mRNA product of a gene being targeted and disrupted so it can’t carry out its normal function which in this case is to produce the BLG protein. The gene knockdown process is carried out by a double stranded RNA molecule (dsRNA) being introduced to the cell that they want to stop the BLG protein made in. The dsRNA is cut into small fragments by a long enzyme called “Dicer”, these small fragments are called interfering RNAs (siRNA). The siRNA bind to special proteins called Argonaute proteins

Cas 9 cleaves the target DNA and creates a Double Strand Break (DSB) that can then be repaired by DNA repair pathway. The primary DNA repair pathway in eukaryotic cells is Non-Homologous End Joining (NHEJ). NHEJ ligates the broken DNA strands together which results in insertions or deletions which lead to gene inactivation. Another DNA repair pathway is Homology Directed Repair (HDR), which is a precise process, usually restricted to dividing cells, that uses exogenous DNA template and allows for desired sequence to be added to the gene (Reis 2014; Wang 2016). The most common result of CRISPR/Cas9 were deletions (Koike 2013). It was observed that HDR was only achieved with simultaneous introduction of repair donor DNA, Cas9, and guide RNAs (Mali Yang 2013). Researchers have altered the domains of Cas9 to get different forms with differing functions. The mutant form Cas9D10A only has nickase activity. This increases the precision of Cas9 by making two Cas9 make a single stranded break to create the double stranded break. This allows for less off target damage because the likely hood of two Cas9 both reacting in off target locations together is rare. This also helps encourage NDR instead of NHEJ. A third Cas9, dCas9, has been developed by mutating the domains of Cas9 to deactivate the DNA cleavage but allow it to remain bound to DNA. This allows blockage of transcription by sterical hindrance, marking DNA in the cell, and adding transcription factors to alter expression (Reis

The process of gene knockdown has been largely unsuccessful so the scientists from AgResearch attempted the process of gene knockdown by RNA interference (RNAi). The role of the RNAi in the nucleus is in the formation of the chromatin domains that help to silence gene expression. First of all the scientists had to identify the gene/s responsible for producing the BLG protein and recognise the base sequence so that the messenger RNA (mRNA) sequence was known. This is done by the restriction enzymes, which cut the DNA at specific places so that the polymerase chain technique can be used to produce many identical copies of the nucleotide sequence. This is done by the Taq polymerase DNA to enlarge the short fragments of DNA. Gene knockdown is a technique where the expression of an organism’s genes is reduced. Gene knockdown by RNA interference (RNAi) silences the genes by mRNA degradation by inserting small interfering RNA (siRNA) into the cytoplasm. Protein-coding genes inside

CRISPR-cas9 works by using a short guide RNA consisting of only 85 bases to precisely direct the protein Cas9 to target sites on the DNA. It then functions as an endonuclease to efficiently produce breaks in the DNA double helix.

RecA is a major DNA repair protein which carries out homologous recombination repair and controls the expression of many other DNA repair proteins in certain bacterial species through the SOS response(48) . The protein coats single-stranded DNA (ssDNA) and causes it to invade double-stranded DNA (dsDNA), thereby catalyzing strand exchange between DNA molecules(49). In addition, RecA is central to the repair response known as SOS mutagenesis (50). The interaction of RecA with ssDNA (resulting from DNA damage) leads to autocatalysis of LexA, which normally inhibits the SOS response(51). While several SOS response enzymes appear to be highly error prone, they nonetheless allow the replication fork to proceed over damaged DNA, guaranteeing DNA