Theme 4: DNA Replication and Mitosis - Module 2: Replication Flashcards
what is the macromolecule that determines the characteristics of the cell?
DNA
what was proposed about the base pairing in DNA
should allow for a mechanism by which the genetic information in DNA could be copied
what did Watson and Crick propose about DNA?
that DNA consists of a pair of template chains which are complementary to each other - prior to replication, hydrogen bonds are broken between complementary strands
what does breaking the hydrogen bonds of complementary strands in DNA allow?
unwinding and separation
what does it mean to say a strand is complementary to another?
each strand contains information necessary to reconstruct the other
what do Watson and Crick believe happens when a cell copies its DNA?
each strand serves as a template for ordering of new nucleotides (accruing to base-pairing rules) into a new complementary strand
what is the end result?
replication of DNA begins with one parent DNA dole helix, the end result would be two new helices
what would be the relation between the new helices and the original?
two new helices, with the new double stranded DNA being an exact copy of the “parental” molecule
what did this model of DNA replication predicted by Watson and Crick propose about when a DNA double helix replicates?
that each of the two daughter DNA molecules would have one old stand from the parental molecule and one newly made strand
what is this model of DNA replication known as?
semiconservative model of DNA replication
what was the conservative fashion DNA replication hypothesis?
two complementary parental strands coming back together after replication
what was the dispersive DNA replication hypothesis?
all four strands combining into a mixture of old and new DNA strands
who’s research conclusively demonstrated that DNA replicates in a semiconservative manner?
Matthew Meselson and Franklin Stahl
what did Matthew Meselson and Franklin Stahl do their research on and in what medium was it done?
- E.coli bacterial cells
- medium containing the nucleotide precursors with radioactively labelled heavy isotope of nitrogen (15N)
what were the bacterial cells transferred into?
medium containing 14N, the lighter isotope
after this point every new strand of replicated DNA would be built containing which isotope?
14N rather than the 15N
what did Meselson and Stahl do throughout their experiment?
extracted DNA samples from the growing bacteria
what did they do with DNA that was extracted from each sample?
centrifuged each sample through a solution that separates the DNA based on differing densities of the radioactively labeled molecules
what does incorporation of the 15N isotope do?
makes the DNA “heavier” than that with the 14N isotope so it ends up near the bottom of the tube
what did Meselsen and Stahl identify about the DNA from the bacteria that had been growing in the media containing the 15N isotope
it had only one distinct band
what was observed after the transfer and then one round of replication in the medium containing the 14N isotope?
DNA also appeared as a single band but with lower density
where was the DNA in the 14N isotope placed in the centrifugation gradient with respect to the 15N isotope?
due to its lower density it was positioned higher than the original 15N band in the centrifugation gradient
what must this band contain?
some hybrid of the 14N and 15N DNA
based on these results why did Meselsen and Stahl reject the conservative model of DNA replication?
no individual distinct band correlated with the “heavier” 15N DNA
what did Meselsen and Stahl do in other to test the other two models?
allowed the bacteria to grow and divide for many generations in the 14N- containing media after transfer from the 15N-containing media
what did they find after many rounds of bacterial replication?
that the extracted DNA could be separated into 2 distinct bands
what were these two distinct bands?
one in the position that contained only 14N and another in the position where the hybrid DNA containing the 15N and 14N would be expected
what were these results consistent with?
the fact that with DNA replication each new double helix is mad dup of one old strand and a new strand
what did this evidence support?
that DNA is replicated in a semiconservative fashion in the cell
what results provide further support for the semiconservative model of DNA replication?
- labelled individual nucleotides with fluorescent labels
- allowed researchers to examine replication of eukaryotic DNA in media containing fluorescent nucleotides and follow entire strands of eukaryotic DNA under the microscope
- found cells could contain hybrid and fully labeled nucleotides
- leads to observed faintly and darkly fluorescing strands of labeled DNA even within one chromosome
is the mechanism of relocation similar in prokaryotes and eukaryotes?
yes
in what phase does DNA replication begin? at what regions along the DNA does replication begin?
- S-phase
- at specific regions along DNA called origins of replication
although prokaryotes and eukaryotes both replicate DNA in a semiconservative manner what leads to a difference in the ways they initiate replication?
organization of their genomes
where does prokaryotic replication begin?
begins at a single origin of replication
what happens after this single origin of replication?
replication continues around the circular chromosome from this one initiation site
what is the process of DNA replication similar to?
transcription
during DNA replication which direction is the template strand copied in? and what direction does is produce its daughter strand?
3’ end to the 5’ end
produces daughter strand that elongates in a 5’ to 3’ direction
during DNA replication what does each incoming complementary nucleotide engage in with the nucleotide template strand? and interacts with what?
hydrogen bond
- interacts with the 3’ hydroxyl of the existing polymer that is forming the new daughter strand
what bond forms between the growing daughter strand and the new incoming nucleotide? what does this allow?
- phosphodiester bond
- allows it to become part of the DNA molecule backbone on the daughter strand ( in the process produces a pyrophosphate)
do the two strands of complementary DNA run in a parallel or antiparalllel fashion?
antiparallel
what do both strands serve as?
simultaneous templates from which DNA can be replicated
what does the unwinding of the DNA double helix result in?
the separation of the parental strands
where does the separation of the parental strands occur?
at regions call replication forks within the origins of replication
what does the initiation of replication require?
a short stretch of an RNA molecule or a primer (usually 5-10 nucleotides long) be synthesized and base pair with the template DNA strands
why is the primer required?
since the enzymatic machinery that elongates a new daughter stand can only do so from an existing piece of DNA or RNA
as elongation continues the polymerization of each newly replicated daughter strand is catalyzed by the DNA polymers enzyme in what direction?
5’ to 3’ direction
what does this enzyme do?
synthesizes a replicated DNA strand from the primers that anneal to the template strand
are there important differences in the manner in which each parent strand is replicated?
yes
what is the leading strand?
only one primer is required and DNA polymerase is able to continuously add nucleotides to the new daughter strand at the replication fork progresses
what type of replication does the other antiparallel parent strand have?
discontinuous (fragmented) replication
what is this produced daughter strand refereed to as?
lagging strand
which end can DNA polymerase add nucleotides to?
the 3’ end of a polymerizing DNA molecule
replication of this strand requires DNA polymerase replicates the DNA in which direction with regards to the replication fork?
away from the replication fork
what is the end result of replication and production of the lagging strand?
lagging strand will contain segments/fragments of DNA
what are these fragments called?
Okazaki fragments
how many primers are required for the leading stand?
one
how many primers are required for the lagging strand?
each Okazaki fragment on the lagging strand is formed by separate primers
what happens after DNA polymerase forms an Okazaki fragment?
another DNA polymerase is able to replace the RNA primer sequences of all daughter strands with DNA nucleotides
have other proteins been found to have important roles during DNA replication?
yes
when various proteins participate in DNA replication what do they form? what is it called?
a single large complex called the replication complex
during initiation what are DNA helices enzymes able to do?
bind to the parental DNA strands at the origin of replication and initiate the unwinding of the DNA double helix
this is accomplished because DNA helices are able to break what?
the hydrogen bonds between complementary nucleotide base pairs
what do newly unwound DNA have the tendency to do?
rejoin
what are single-stranded binding proteins used for?
bind to and stabilize each parental strand until elongation can begin
a third family of proteins, the topoisomerases do what?
able to bind upstream of the replication fork
what do topoisomerases do?
minimize the torsional strain
why is there torsional strain?
brought about from the unwinding that occurs at the replication fork
what do topoisomerase proteins serve as? what do they trigger?
initiator proteins that trigger the process of unwinding the origin of replication
what process follows this?
process of primer synthesis
what does this mark the beginning of?
the synthesis of the new daughter DNA molecules
what is the name of the RNA polymerase enzyme that is used to synthesize the short RNA stretches of nucleotides which are complementary to the parental strands from which DNA polymerase can then elongate from?
RNA primase
which DNA polymerase does most of the elongation work in prokaryotes?
DNA polymerase 3
what is DNA polymerase 1 responsible for?
is the enzyme that is responsible for removing the RNA primer after DNA replication and replacing those short sequences with DNA nucleotides
most of the replication process between eukaryotes and prokaryotes is similar, however what is a difference?
eukaryotes utilize a different set of DNA polymerases
does synthesis of the replicated leading and lagging daughter strands occur simultaneously?
yes
why is the lagging strand named that?
due to the delay in synthesis brought about relative to the leading strand
when can each new fragment of the lagging strand be replicated?
when enough of the template DNA is revealed at the replication fork
what does the lagging strand also require?
some post-replication processing
what happens in prokaryotes since they have one origin of replication?
during replication of their circular DNA, the excised primer is replaced by specific DNA nucleotides and there is no gap in the newly synthesized DNA
is it the same in eukaryotes?
no
the replacement of the RNA primer with DNA nucleotides leaves what?
a sugar phosphate backbone at the 3’ end with a free phosphate backbone
where is this prevalent?
along the Okazaki fragments of the lagging strand
what is the name of the enzyme that is able to join the 3’ end of a fragment to an adjacent DNA nucleotide
DNA ligase
how is this done?
by catalyzing the phosphodiester bond formation along this region of the DNA backbone
what happens as a result?
joins adjacent replicated Okazaki fragments together
can the specific manner in which DNA is replicated from a parent template strand be solely attributed to the specificity of base pairing?
no
can errors in DNA replication occur?
they are rare, but yes
when can these errors occur?
during the initial pairing between incoming nucleotides and those of the template strand
does the cell have an innate proofreading mechanism?
yes
during DNA replication, what does DNA polymerases do?
able to proofread each added nucleotide relative to the template strand, as each DNA nucleotide is added to the growing daughter strand
what happens if an incorrect nucleotide pairing is detected?
DNA polymerase removes the incorrect nucleotide, the correct nucleotide is added and then replication can continue by the DNA polymerase
is it possible for other enzymes to help with the correction of replication errors?
yes
what have we seen with regards to prokaryotic replication?
replication begins at one origin of replication, after which replication continues around the circular chromosome from the one initiation site
where does eukaryotic replication originate?
originates at many origins of replication along the linear chromosomes
despite the differences in the number of origins of replication, do prokaryotes and eukaryotes share many common features during replication?
yes
what is common among replication in prokaryotes and eukaryotes?
- both require a primer for initiation of replication to occur
- elongation is always in the 5’ to 3’ direction
- specialized proteins are utilized within a replication complex which allows for the replication of daughter strands from the parental DNA template strands
- both have a leading and lagging strand
DNA replication in eukaryotes leads to a leading strand that replicates what?
the whole template strand
is this is the same with the lagging strand?
no
what is the DNA replication machinery not able to do in eukaryotes due to its linear shape?
has no way to complete the 5’ ends of daughter strands
what happens when an RNA primer on the lagging strand is bound to the very end of a template strand?
once that RNA primer is removed, it can not be replaced with DNA nucleotides because there is no 3’ end available for nucleotide addition and phosphodiester bond formation
as a result what is the result of repeated rounds of DNA replication?
shorter and shorter DNA molecules are produced with uneven ends
why is this a problem for eukaryotic chromosomes but not prokaryotic chromosomes?
because eukaryotic chromosomes are linear where as prokaryotes have circular chromosomes
does this shortening cause a problem in eukaryotes?
no
what are the regions at the ends of linear eukaryotic chromosomes called?
telomeres
what are telomeres?
special nucleotides sequences that are mainly made up of repetitions of one short nucleotide sequence
what do human telomeres contain?
characteristic six-nucleotide sequence TTAGGG repeated between hundreds to thousands of times - leads to many tandem repeats of G-T rich sequences
what does this non-coding repetitive sequence of nucleotides serve as?
a buffer zone
what does the buffer zone do?
protects coding genes within chromosomes
what happens to the size of telomeres during successive rounds of replication?
they become shorter
therefore what can we imagine about telomeric DNA in the cells of older individuals?
telomeric DNA would be shorter in dividing cells of older individuals
what cells is this the case for?
somatic cells
would it be a problem if the chromosomes of germ cells became shorter with each cell cycle?
yes
what would happen if this were the case?
lead to production of gametes with missing information
where else would this be a problem?
in embryonic stem cells that must go through many many rounds of replication to produce fully developed organisms
where does telomere shortening not occur?
gametes or in stem cells
what is unique about these types of cells?
they have a special telomerase enzyme which catalyzes the lengthening of telomeres in these eukaryotic cells
what is telomerase a specific type of?
reverse transcriptase
what is this enzyme able to do?
synthesize DNA from an RNA template
what is the telomerase?
a ribonucleoprotein that contains the RNA template as part of the complex itself
what does the RNA template of the telomerase serve an important role in?
elongating the linear chromosomes of stem cells and germ cells
to elongate the telomere regions what does the telomerase do?
binds to the tail of the telomere and subsequently catalyzes the extension of the template strand by adding telomere repeats
what happens once the telomerase has extended the template DNA strand?
primase, DNA polymerase and ligase are able to go back and complete the daughter strand replication from the reminder of the template strand
