- Why are mutations at D185, D186 and D110 are so detrimental to RT activity? (15 Points)
Mutations at D185, D186 and D110 are very detrimental to RT activity owing to the fact that the three carboxylate residues have been documented to participate in some forms of physiological processes and metal coordination function that is fundamentally required in the binding of deoxyrobonucleotide substrates to the enzyme. And so, when these subunits are interfered as a result of mutation then the fore mentioned biological process are essentially incapacitated. Furthermore, the region was also subcloned due to mutagenesis hence the high expression vectors are essentially covered and are not fundamentally expressed. The enzyme activities on the subunits have been incapacitated leading to detrimental reconstitution to RT activity.
2. The Harris et al. paper studied different mutations in the dNTP-binding pocket and active site of HIV-1 RT and their affect on fidelity. Why is studying fidelity so important with this enzyme? (15 Points)
The Dntp binding pocket of the active site of HIV-1 Reverse Transcriptase type 1 and DNA polymerase are usually labeled using a photoreactive analog of Dctp. DNA polymerase ought to identify and incorporate the right Dntp with potentially high fidelity to sustain the genetic stability hence its importance in the whole enzymatic activities. Since the enzyme has to bide to an infinite number of DNA sequences during the replication process then it goes without say that association between polymerase and the DNA primer is fundamentally non-specific. In essence the fidelity of a given DNA polymerase is what contributes to an accurate replication of a desired template. According to Harris et al. on study of different strains of mutations in the Dntp-binding pocket and their effect on fidelity works to illustrate effective discrimination of appropriate and inappropriate nucleotide incorporation activity referred to as proofreading.
The study of fidelity by a couple of researchers is in essence of great importance owing to the fact that fidelity plays an integral role in biological processes in which DNA sequences must be accurate after an amplification process. One common enzymatic process is subcloning DNA for protein expressions.
3. What is the reasoning behind Rezende's statement that "M184I creates significantly more frame shift mutations outside homopolymericruns." (15 Points)
The reason behind Rezende’s statement that M1841 creates more frame shift mutations owing to the fact that M184V is essentially reduces susceptibility to the drugs up to over 100 fold. M184V results in low resistance levels in ABC and ddl; it also cause increased susceptibility to AZT, d4T and TDF while at the same time slowing the emergence of AZT and TDF resistance with remarkably low viral replication. These activities of M184I essentially emerges before M184V as it works with a common HIV-1 nucleotide substitution and this requires significantly more frame shift mutations outside homoplymericruns as construed by Rezende’s statement.
However M184V is more aggressive than in the first couple of weeks of viral replication though with both have similar resistance profiles.
4. Explain how a pale blue mutant (color score of 1) is created over a sky blue mutant (color score of 2) in the Forward Mutation Assay. (15 Points)
Mutation can essentially occur in different directions and these include forward mutation, true reversion and suppressor mutation. The forwarded mutation attributed to essentially works to change a wild type allele into a new allele for instance a change from a normal allele to an albino allele
In this case a sky blue mutant found in the natural population referred to as the wild type has been exposed t a forward mutation leading to a pale blue mutant that deviates from the wild type to mutant form.
5. (i) In the Scott paper on primer-rescue, Figure 1B showed products being formed, albeit very slightly in the absence of RT and the pyrophosphate donors. Explain this result. (ii) When three of the four components was added, you lost a good concentration of the primer, why is that? (20 Points)
Products can be formed though at a reduced rate owing to the absence of RT and the pyrophosphate donors. In vitro conditions, ATP can serve in the absence of pyrophosphate with the incorporation of AZT containing therapy resistance. ATP can essentially serve as a pyrophosphate donor for the primer unblocking RT (ter).
Good primer concentration was lost because of inhibition processes from components added.
6. Why did Scott and his team use the Klenow fragment for the extension of the rescued primer, if RT could easily polymerize from the open primer? (20 Points)
Klenow fragment is more flexible in the extension process of the rescued primer.
Klenow fragment was used by Scott also because of the fact that it is isolated from a recombinant source and essentially generates using random primers. This is not the case with other open primers.
Exposure of DNA polymerase I to protease subtilisin reduces the compound to small fragment but retaining 5’-->3’ exonuclease activity and a larger segment on the DNA polymerase compound called Klenow fragment widely used in molecular processes and the same was used by Scott and team for extension of rescued primer.
Explain Harris's hypothesis as to why RT has such high fidelity. Be very specific and give details.
RT exhibits a significantly high fidelity rate than other known DNA polymerases since it introduces errors at a remarkably high frequencies of up to 1,500 to 30,000 nucleotides during the process of cDNA processing. They are essentially the core initiators of errors in RT-PCR with the ultra fidelity acting as the PCR enzyme. The errors postulated at this stage are amplified during transcription hence the need to correct the numerous errors after RT-PCR.
