DNA Replication I


so we left off with the study of DNA
structure and we mentioned the aesthetic beauty of the molecule and indeed it is a pretty molecule, and part of it’s aesthetic beauty comes from the way its structure of the molecule allows for its
replication with fidelity and you heard discussing that feature in that movie clip
of people which described the structure of DNA immediately recognized that the molecule provides the means for its accurate reproduction in taking up a parental molecule and producing two daughters molecules which would be the requirement
of the genetic material and that replication process could be described as semi conservative in that given the complementarity of the basis in
opposite strands of a double helix if the strands separated so we separated
this blue strands and then build gray strands upon the each blue strand using the base complementarity using a strand to build a strand which is complementary to the original strand in
its base pair sequence a is paired with ts g’s paired with c’s then you would end up with two daughter molecules that are were identical in the nucleotide sequence to the parental
molecule now Watson and Crick immediately realized that this would be
a very effective means for replication of the double helix but for later other
possibilities you could imagine all the mechanistically a little bit difficult
to imagine what you could say that a parental DNA molecule remains and a new tire new
double helix is synthesized using the information present in the original
double helix to produce an identical daughter double helix or you could imagine that would be conservative model of replication imagined dispersive model of replication
in which part of the new daughter molecules the daughter double helixes will be part of each strand would would
be synthesized in new and parts would be remains from the original parental helix this but this semi conservative
model in which one the parental strains is completely conserved each daughter and as has served as a template
upon which to synthesize a daughter strands this which model seems most appealing and neselson and stall did a very famous experiment one of the best I think in all of molecular biology, in which they demonstrated creeks proposed somewhat conservative
model of DNA replication is in fact correct. So here’s what they did they labeled, they used bacteria Ecoli the human gut bacteria bacteria in media in liquid medium that
contained the heavy isotope of nitrogen N15 and then that would label their DNA because of the nitrogenous bases because of the heavy nitrogen and they could then switch the Ecoli to the medium which contained the normal isotope of nitrogen, its not radioactively labeled and the bacteria as the day progressed in
replicated incorporate light nitrogen into their nitrogenous bases and therefore into their DNA and then they could take take samples out of this culture in
which he labeled DNA containing DNA level with the heaviest of nitrogen was transferred to a medium which contained a light isotope of nitrogen they could take samples at various times and centrifuge
the isolate DNA and centrifuge that DNA in an super centrifuge at very high speeds and at those high speeds you can separate out molecules that have greater mass than
other molecules so completely labeled DNA with sediment more towards the bottom of their centrifuge tube that would
this interviews to that would completely like DNA so they could determine with
the positions of sedimentation molecules in bacteria that were sampled at various times after they had been transferred to the light medium and could detect the labeled DNA by exposing
the centrifuge tubes to photographic film and determine this banding pattern
which describes the position of labeled radioactive DNA so at time 0 as soon as you transfer
the bacteria into this new medium they still have labeled heavy DNA both strands of the double helix would
be level with radioactive nitrogen N15 and indeed if we go top to bottom this we put the centrifuge tubes horizontally top bottom indeed the heavily labeled DNA at time 0 produces a single band that sediments towards the bottom of the centrifuge tube but after one round of DNA replication what you find is another single band of labeled DNA but this sediments and a
lighter and a position that would correspond to lighter doublehelix with less mass than
both strands label and this strongly implied that after one generation one
round of DNA replication in other words there was one type of DNA double helix
produced which was a hybrid between light newly synthesized DNA and parental labeled DNA that at the end fifteen isotope so we detect the label and a position that is has sediments higher position of centrifuge tube than centered DNA two rounds of DNA replication they
observed two DNA bands contained radioactive DNA what one was actually this is not
labeled DNA they are detecting all all DNA here they are putting a dye in their tube it allows them to visualize visualized DNA, all DNA and after ten rounds of replication what you find is that and a completely light band of DNA and this sense if you consider the semi
conservative model of DNA replication after one round you should a hybrid molecules
and after two rounds when this molecule that is hybrid that is doubt that is has won
parental heavily labeled strand and one completely new light strand when that molecule replicates in the light medium in bacteria within the light medium and the light strands shown in gold here would serve as a template upon which to synthesize a new unlabeled strand whereas the original strand of DNA that came from the parent here would be in fact the template upon which a new strand would be synthesized in the world would have a hybrid band of DNA and that is
exactly what we see here are two bands small experiment was an elegant way to
shown that this semi conservative model of replication replication was um most likely the
correct one but you will note that after one generation the hybrid molecule predicted by the semi conservative model replication produces one completely heavy strand and one completely light strand now you would’ve obtained a hybrid density molecule if the dispersive model where correct after one
generation you would have hybrid molecules that would have density intermediate between completely light DNA completely heavy DNA but if you consider what the dispersive
model would produce in the next generation since every strand here is a hybrid between labeled and
unlabeled DNA in the next generation you would still have hybrid molecules you
would not completely like double helix produced and generation to using the
dispersive so their results in the second generation confirmed that the
semi conservative model is the correct in that day if each parental strand serves as a template upon which each model synthesized then this hybrid molecule in the semi conservative mode would produce another molecule in the second generation and one completely unlabeled molecule and after Watson and crick mezelston and crawl solve the structure of DNA show that semi conservative replication was the mode of DNA replication and soon became obvious but we soon discovered replication of bacterial chromosomes would proceed
preceded by directionally from origin to a termination point of about 100 degrees opposite on
the bacterial circular chromosome so here we have two strands of DNA
indicated in purple here and here we schematically show that there aren’t two directions of
replication emanating from the origin of replication and new daughter strands
are being shown in red using the contact template information present the parental purple strength in both
directions bidirectional replication in other words
and we referred to the each of these points of replication when moving in this
direction and one moving in that direction referred to these replication forks the
replication forks and chromosomal replication forks proceed round with the
enzymatic structures required to synthesize DNA there are enzymes of course responsible for synthesize of new DNA strands that is the polymerization of
nucleotides into new strands of DNA each direction and those enzyme complexes proceed around the chromosome until the termination point ultimately yielding then two molecules each of which contains completely conserved parental strand and purple and a completely new synthesized strand shown in red but bacterial chromosomes strands are relatively small compared to eukaryotes and as we’ve seen bacterial chromosomes are circular eukaryotic chromosomes are linear not circular if we look at the replication of eukaryotic chromosomes
electron micrograph replication and replication in eukaryotic chromosomes and what im circling here are replication forks and these are proceeding bidirectionally from up more points of origin somewhere in the middle here and here is
a replication port received by directional and there is another one and
here’s another one and there we can get this electron micrograph as follows multiple electron we have multiple
replication forks rather for other and and this replication forks are then proceeded
then bidirectionally and then that you have multiple sites of regions of DNA replication there are multiple origins and cites of DNA replication in the eukaryotic replication and many hundreds replication perceives bidirectionally from a given location now the next part of this lecture we’re going to examine what happens at the replication fork kind of the enzymology of DNA replication and look at the way that new nucleotide are inserted into growing strands Built up from the information present in the parental strands of DNA and the way daughter helixes are produced

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