A scientist is replicating human DNA in a test tube and has added intact DNA, the replisome complex, and the four deoxyribonucleoside triphosphates. To the surprise of the scientist, there was no DNA synthesized, as determined by the incorporation of radio-labeled precursors into acid-precipitable material. The scientist’s failure to synthesize DNA is most likely due to a lack of which of the following in his reaction mixture?
A. Reverse transcriptase
B. Ribonucleoside triphosphates
D. Dideoxynucleoside triphosphates
E. Sigma factor
The answer is B: Ribonucleoside triphosphates.
DNA Replication is the basis for inheritance. It is the fundamental process occurring in all cells for copying DNA to transfer the genetic information to daughter cells. Each cell must replicate its DNA before division.
DNA polymerase, the main enzyme for polymerization of DNA, cannot initiate DNA synthesis de novo. It can only add nucleotides to 3′ end of a growing DNA strand, it needs a “starter” nucleotide to make a bond. A Primase initiates synthesis of a primer, an RNA molecule that is essential for priming DNA synthesis (the primers are composed of ribonucleotides)- See figure-1.Primer serves as a starter sequence for DNA polymerase. RNA Primer has a free 3’OH group to which the first nucleotide is bound (figure-1).
Figure-1- A short RNA fragment is needed by DNA polymerase to add nucleotides. The new nucleotide is added to the 3′-OH group of the primer forming 3′-5′ phosphodiester linkage.
Only one RNA Primer is required for the leading strand. Multiple primers are needed for the lagging strand (figure-2).
Figure-2- Number of primers needed depends upon the number of okazaki fragments.
The components required for replication and the steps involved are shown in the figure below (figure-3).
Figure-3- Steps of DNA replication. The process starts with the unwinding of DNA by helicase.The single stranded structure is maintained by single strand binding proteins (SSB). DNA polymerase adds nucleotides to the RNA primer. Leading strand is continuously synthesized, while the lagging strand is synthesized in a discontinuous manner. The primers are removed and nicks are sealed by DNA ligase.
In the given situation,
The scientist has added all the enzymes needed for replication (The replisome is a complex containing all the proteins and enzymes needed for replication)- see figure-3 .
Reverse transcriptase is not required in the given situation. It is an enzyme for the synthesis of DNA from RNA, It is RNA dependent DNA polymerase.
The DNA fragment to be replicated has been added. Each DNA strand serves as a template to guide the synthesis of complementary strand.
Dideoxynucleoside triphosphates are not needed for DNA replication, in fact they act as inhibitors of DNA replication by not providing fee 3′-OH group for further polymerization.
Sigma factor is also not the right option, it is a subunit of RNA polymerase and is required for promoter identification in prokaryotic transcription. It is not required for DNA replication.
Hence the right option is B- Ribonucleoside triphosphates
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