Base Excision Repair Essay

ends when the RNA polymerase reaches a triplet of bases then the DNA molecules re-

This tube contains DNA, forward and reverse primers, Taq DNA polymerase, and deoxy nucleotides. After removing the tube from the thermocycler and letting the tube cool, the primers will begin base-pairing with the DNA. The polymerase will then extend the primers and this will complete the first cycle of the reaction causing 2 copies of the DNA to be produced. This cycle can be repeated 30-35 times, which can create 1 billion copies of a segment of DNA (Dulai,

In order to analyze the DNA better in this lab we will need to split the sugar-phosphate bond between the nucleotide bases. Naturally, DNA has the structure of a double helix or two strands intertwined but as we add enzymes the DNA will break in the process of hydrolysis. These enzymes, most commonly EcoRl and Pstl are restriction endonucleases. As we know,

Figure 2. Schematic illustration of generating Nf1-/- iPS cells from its Nf1+/- counterparts. (A) is the genotype of Nf1+/- iPS cells. (B) and (C) are two kinds of Nf1-/- iPS cells after Crispr/cas9 mediated NF1 genome editing in Nf1+/- iPS cells. In the presence of the repair template, Crispr/cas9 is able to introduce a nonsense mutation (c.94-96>TAA) into the exon 2 of NF1 genome via the homology directed repair (HDR). Without the repair template, random insertions and/or deletions may be introduced by non-homologous end joining (NHEJ). Nf1-/- iPS cells are to be generated by introducing

possible error allowing repair thus achieving high fidelity in transcription. Also, the DNA damage response system can activate checkpoints inducing cell cycle arrest, allowing time for different mechanisms such as Base, Nucleotide Excision Repair and Mismatch Repair system which, involving specialized proteins, will excise and repair the incurred error.

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.

This is essentially energetically neutral (no need for ATP hydrolysis) and results in the formation of a free 3' hydroxyl group and a 5' phosphate group on the same strand. The reactive hydroxyl group is positioned by the recombinase to attack the phosphodiester bond of opposite strand, forming two DNA ends: a hairpin (stem-loop) on the coding segment and a blunt end on the signal segment.[14] The current model is that DNA nicking and hairpin formation occurs on both strands simultaneously (or nearly so) in a complex known as a recombination

Eukaryotic chromosomes have many origins of the replication. Each origin of replication form the duplicate DNA on the side of the origin of the replication. Once the leading strand of the duplicate DNA strnd reaches the lagging strand of the another duplicate one and the lagging strand will go through the 5’ end of the okazaki fragment.okazaki fragment is in the same bubble. DNA polymerase enzyme will go through the already replicated DNA template but it can’t be stop the formation of phosphodiester bond.it forms between the two DNA new strand.so the new DNA can’t be attached and ithis full replicated DNA strand called as nicks. Once all the templates are replicated it’s not means the replication process is over.

To repair mismatched bases, the system has to know which base is the correct one. In order to recognize the parental strand, the characteristic than can be detect isi it is a DNA strand that has been methylated. Once in a while, there will be times when the polymerase would accidentally place the wrong base across the template DNA strand during the replication of DNA. Usually it would detect it mistakes, and correct itself. But, if polymerase failed to fix its mistakes, there are some types of repair enzymes that would scan the DNA strand and proof reed the strand again. However, there might still be a wrongly placed base pair of the new strand compare to the parental strand. Figure 7 in page 15 show the pathways

Once such break is generated, the inherent DNA repair machinery of the cell is activated, and during the repair process of double-strand break the genome is modified.

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

After DNA damage, due to the accumulation of mismatch repair, it causes genomic instability. Besides, the genetic information is changed. P53 can participate in DNA repair process, which itself has a DNA-binding domain nucleic acid endonuclease activity, resectable mismatch nucleotide, nucleotide binding and regulate endo repair factor XPB and XPD activity, affect its DNA recombination and

How are these breaks repaired? DSBs can be repaired through distinct mechanistically set pathways, as shown in figure 1, such as Homologous recombination repairs (HRR), Non-end joining homologous repair (NHEJ) and more recently discovered Microhomology-mediated end joining (MMEJ). HRR uses a homologous template to accurately repair the broken strand, whereas NHEJ relies on Ku proteins (Fnu et al., 2011) and DNA ligase to repair DNA double strands, often times causing small insertions or deletions at the joining site. MMEJ, a more recent finding, uses a 5-25 bp micorohomologous sequence to align the broken ends simultaneously always causing deletions in the previously broken strand (McVey and Lee, 2008).

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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