MBI- Replication Flashcards
what is semuconservative type of dna replication
The scheme suggested that the two strands would
separate and act as a template for the synthesis of new
complementary strands. After the completion of
replication, each DNA molecule would have one
parental and one newly synthesised strand. This
scheme was termed as semiconservative DNA
replication
describe meselson and stah’s expt
(i) They grew E. coli in a medium containing 15NH4Cl (15N is the heavy
isotope of nitrogen) as the only nitrogen source for many
generations. The result was that 15N was incorporated into newly
synthesised DNA (as well as other nitrogen containing compounds).
This heavy DNA molecule could be distinguished from the normal
DNA by centrifugation in a cesium chloride (CsCl) density gradient
(Please note that 15N is not a radioactive isotope, and it can be
separated from 14N only based on densities).
(ii) Then they transferred the cells into a medium with normal
14NH4Cl and took samples at various definite time intervals as
the cells multiplied, and extracted the DNA that remained as
double-stranded helices. The various samples were separated
independently on CsCl gradients to measure the densities of
DNA
result of meselson’s and stahl’s expt
Thus, the DNA that was extracted from the culture one
generation after the transfer from 15N to 14N medium [that is
after 20 minutes; E. coli divides in 20 minutes] had a hybrid or
intermediate density. DNA extracted from the culture after
another generation [that is after 40 minutes, II generation] was composed of equal amounts of this hybrid DNA and of ‘light’
DNA.
what were some similar expts conducted
Very similar experiments involving use of radioactive thymidine to
detect distribution of newly synthesised DNA in the chromosomes was
performed on Vicia faba (faba beans) by Taylor and colleagues in 1958.
The experiments proved that the DNA in chromosomes also replicate
semiconservatively
describe the function of DNA polymerase
The main enzyme is referred to as DNA-dependent
DNA polymerase, since it uses a DNA template to catalyse the
polymerisation of deoxynucleotides. These enzymes are highly efficient
enzymes as they have to catalyse polymerisation of a large number of
nucleotides in a very short time.( 2000 bp mer minute)
ii)Not only do
these polymerases have to be fast, but they also have to catalyse the reaction
with high degree of accuracy. Any mistake during replication would result
into mutations.
iii)The DNA-dependent DNA
polymerases catalyse polymerisation only in one direction, that is 5’à3’.
what are replication forks
For long DNA molecules, since the two strands of
DNA cannot be separated in its entire length (due to very high energy
requirement), the replication occur within a small opening of the DNA
helix, referred to as replication fork.
The uncoiling of DNA results in the creation of y shaped regions called replication forks
where is the energy for replication coming from
Furthermore, energetically replication is a very expensive
process. Deoxyribonucleoside triphosphates serve dual purposes. In
addition to acting as substrates, they provide energy for polymerisation
reaction (the two terminal phosphates in a deoxynucleoside triphosphates
are high-energy phosphates, same as in case of ATP).
what is the continuous replication and discontinuous replication
The DNA-dependent DNA
polymerases catalyse polymerisation only in one direction, that is 5’à3’.
This creates some additional complications at the replicating fork.
Consequently, on one strand (the template with polarity 3’à5’), the
replication is continuous, while on the other (the template with
polarity 5’à3’), it is discontinuous. The discontinuously synthesised
fragments are later joined by the enzyme DNA ligase
can dna polymerase initiate a dna chain
It cannot initiate, it can only elongate a strand. It has to attach them onto the end of a newly synthesized daughter strand. Hence it needs a primer to synthesize a short strand to which it will elongate.
- A primer is a short polynucleotide segment that primes, or prepares, the way for DNA replication by helping DNA polymerase to get started in doing its job. The primer is made out of RNA, so it’s called an RNA primer. The job of RNA primase is to make, or synthesize, a primer for replication to start.
what are origins of replication
Also the replication does not initiate randomly at any place
in DNA. There is a definite region in E. coli DNA where the replication
originates. Such regions are termed as origin of replication.
when does dna replication take place
In eukaryotes, the replication of
DNA takes place at S-phase of the cell-cycle. The
replication of DNA and cell division cycle should be
highly coordinated.
