DNA replication Flashcards
What are the three possible mechanisms for DNA replication? Briefly describe each of the three and state
which is the mechanism employed in living systems
Conservative: parental double helix remains intact (is totally conserved) and directs the synthesis of a “progeny”
double helix composed of two newly synthesized strands.
Semi-conservative: the DNA strands separate and each separated strand acts as a template for DNA replication
such that each new copy of the DNA contains one template strand and one newly synthesized strand.
Dispersive/distributive: here, the parental strand and progeny strand become interspersed such that both strands
contain both template and newly synthesized DNA
Living systems employ a semi-conservative mechanism of DNA replication
What is the Klenow fragment of a prokaryotic polymerase and what functions does it carry out
The Klenow fragment is the large C-terminal fragment of prokaryotic DNA polymerase I which has both 5’-3’
polymerase and 3’-5’ exonuclease activity, the latter of which carries out a proof-reading function during DNA
replication.
RFC
Replication factor C -this is the clamp loader, equivalent to sub-unit γ of prokaryotic DNA polymerase III
RPA
Replication Protein A -Replication Protein A – binds single-stranded DNA at the opening of the replication fork, equivalent to SSB (single
stranded DNA binding protein) in prokaryote
PCNA
Proliferating cell nuclear antigen – this is the clamp for the DNA polymerase, equivalent to the β sub-unit of
prokaryotic DNA polymerase III
What does the term processivity refer to and why is this important in DNA replication?
The ability of an enzyme to catalyze many reactions before releasing its substrate is called processivity. During
DNA replication, the addition of thousands of nucleotides must be catalyzed before the polymerase can release the
template in order for the process to be time and energy efficient. This is due to the polymeric nature of DNA and
the large size of chromosomes.
Explain the end replication problem as described by Watson and how this process is overcome in longlived cells
Because DNA polymerases act in a 5’-3’ direction and can only add nucleotides to an existing free 3’ OH group, this
means that cells cannot replicate the extreme 5’ ends of chromosomes on the lagging strand and they would get
shorter after each cell division. In particular, the replacement of the RNA primers of Okozaki fragments by DNA
polymerase I requires the 3’ OH group of an adjacent nucleotide and ligase is only capable of joining two existing
nucleotides together. Long-lived cells use telomerase to overcome this problem. Telomerase is composed of protein
(reverse transcriptase) and RNA complementary to the telomere repeat. The RNA binds to the terminal telomere
repeat and reverse transcriptase catalyzes the addition of new repeats. In this way, telomerase compensates by
lengthening the 5 to 3 strand to provide a primer binding site that allows the subsequent extension of the 3 - 5
strand. Primase can then extend the lagging strand and any genetic material that may be lost in subsequent
rounds of replication is non-essential telomeric repeat sequences.
What are the three steps of the PCR and what happens in each of these steps
Denaturation: separation of the two DNA strands at high temperature (92-95ºC)
Annealing: attachment of primers to template sequence(50-65ºC)
Extension: synthesis of new DNA strands by polymerase (72ºC)
Explain how the discovery of Taq polymerase revolutionized the application of the PCR
This is a thermostable polymerase (from a thermophilic bacterium) that is capable of surviving the high
temperature of the denaturation step such that it is not necessary to replace the polymerase after each step. This
allowed for the automation of PCR and made the technology more feasible and affordable.
What is meant by the fidelity of a DNA polymerase?
the rate of error of polymarase
What is the difference between DNA damage and mutation?
DNA damage is a change in the chemical structure of DNA eg. A break in the DNA strand, a change in the structure
of a nitrogenous base, a missing nitrogenous base from a nucleotide. A mutation, on the other hand, is a heritable
permanent change in the sequence of bases and cannot be repaired.
How do bacteria distinguish between template (correct) strand and daughter stands during DNA repair?
Bacteria are able to methylate their DNA, thus the template strand is methylated on the adenine base within a
GATC sequence
What are intercalating agents and how do they cause DNA damage? Give an example
These are molecules whose dimensions are roughly the same as those of a purine-pyrimidine pair and are able to
insert themselves between two base-pairs in a DNA molecule. This may result in misalignment of the base pairs
resulting in a frameshift mutation by either insertion or deletion of a base
Describe what is meant by the deamination of nitrogenous bases during DNA damage and what are the
resultant bases?
This is the removal of the amino group from a nitrogenous group. Cytosine is converted to Uracil , Guanine to
Xanthine and Adenine to Hypoxanthine.
What is the mechanism of DNA repair by which only the damaged bases are repaired? What are the two
enzymes involved?
Base excision repair. Carried out by the enzymes DNA glycosylase and AP endonuclease
