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COMPREHENSION QUESTIONS
1.
*2.
How did Meselson and Stahl demonstrate that replication in E. coli takes place in a
semiconservative manner?
Meselson and Stahl grew E. coli cells in a medium containing the heavy isotope of
nitrogen (15N) for several generations. The 15N was incorporated in the DNA of the
E. coli cells. The E. coli cells were then switched to a medium containing the
common form of nitrogen (14N) and allowed to proceed through a few cycles of
cellular generations. Samples of the bacteria were removed at each cellular
generation. Using equilibrium density gradient centrifugation, Meselson and Stahl
were able to distinguish DNAs that contained only 15N from DNAs that contained
only 14N or a mixture of 15N and 14N because DNAs containing the 15N isotope are
heavier. The more 15N a DNA molecule contains, the further it will sediment
during equilibrium density gradient centrifugation. DNA from cells grown in the
15
N medium produced only a single band at the expected position during
centrifugation. After one round of replication in the 14N medium, one band was
present following centrifugation, but the band was located at a position
intermediate to that of a DNA band containing only 15N and a DNA band
containing only 14N. After two rounds of replication, two bands of DNA were
present. One band was located at a position intermediate to that of a DNA band
containing only 15N and a DNA band containing only 14N, while the other band was
at a position expected for DNA containing only 14N. These results were consistent
with the predictions of semi-conservative replication and incompatible with the
predictions of both conservative and dispersive replication.
*3.
4.
5.
Origin
Leading Strand
Lagging Strand
Okazaki
Fragments
3'
5'
3'
5'
3'
5'
3'
5'
5'
3'
3'
5'
Unwinding
5'
3'
3'
Unwinding
Origin
Leading Strand
Lagging Strand
Okazaki
Fragments
RNA Primers
6.
*7.
8.
List the different proteins and enzymes taking part in bacterial replication. Give the
function of each in the replication process.
DNA polymerase III is the primary replication polymerase. It elongates a new
nucleotide strand from the 3'OH of the primer.
DNA polymerase I removes the RNA nucleotides of the primers and replaces
them with DNA nucleotides.
DNA ligase connects Okazaki fragments by sealing nicks in the sugar
phosphate backbone.
DNA primase synthesizes the RNA primers that provide the 3'OH group
needed for DNA polymerase III to initiate DNA synthesis.
DNA helicase unwinds the double-helix by breaking the hydrogen bonding
between the two strands at the replication fork.
DNA gyrase reduces DNA supercoiling and torsional strain that is created
ahead of the replication fork by making double-stranded breaks in the DNA and
passing another segment of the helix through the break before resealing it. Gyrase
12.
How does replication licensing ensure that DNA is replicated only once at each
origin per cell cycle?
Only replication origins to which replication licensing factor (RPF) has bound can
undergo initiation. Shortly after the completion of mitosis, RPF binds the origin
during G1 and is removed by the replication machinery during S phase.
*13. In what ways is eukaryotic replication similar to bacterial replication, and in what
ways is it different?
Both eukaryotic and bacterial replication of DNA replication share some basic
Outline in words and pictures how telomeres at the end of eukaryotic chromosomes
are replicated.
Telomeres are replicated by the enzyme telomerase. Telomerase, a
ribonucleoprotein, consists of both protein and a RNA molecule that is
complementary to the 3' end of the DNA of a eukaryotic chromosome. The RNA
molecule also serves as a template for the addition of nucleotides to the 3' end.
After the 3' end has been extended, the 5' end of the DNA can be extended as well,
possibly by lagging strand synthesis of a DNA polymerase using the extended 3'
end as a template.
DNA replication of the linear eukaryotic chromosomes generates a 3'
overhang. Part of the RNA sequence within telomerase is complementary to the
overhang.
TELOMERASE
5'CAACCCCAA
3'
5'CCCCAA
3'GGGGTTGGGGTT
Toward Centromere
Telomerase RNA sequence pairs with the 3' overhang and serves as a template
for the addition of DNA nucleotides to the 3' end of the DNA molecule, which
serves to extend the 3' end of the chromosome.
TELOMERASE
5'CAACCCCAA
3'GGGGTTGGGGTT
TELOMERASE
5'CCCCAA
5'CAACCCCAA3'GGGGTTGGGGTTGGGGTT
PRIMASE
CCCCAACCC
5'CCCCAA
3'GGGGTTGGGGTTGGGGTTGGGGTTGGGGTT
RNA PRIMER
DNA POLYMERASE
5'CCCCAACCC CAACCCCAACCCC
CCCCAA
3'GGGGTTGGGGTTGGGGTTGGGGTTGGGGTT
15. Briefly outline with diagrams the Holliday model of homologous recombination.
Single strand breaks in two adjacent DNA molecules allows for the free ends of the
two strands to invade the other DNA molecule. The invading strand joins to the
broken end of the other strand displacing complementary base pairs, which allows
for the formation of a cross bridge.
After formation, the cross bridge can move along the DNA molecules in a
process called branch migration. The migration creates a structure called the
Holliday intermediate. Redrawing the junction allows us to see how the cleavage
might take place to generate recombinant DNA molecules.
*16. What are some of the enzymes taking part in recombination in E. coli and what
roles do they play?
(1) RecBCD protein unwinds double-stranded DNA and can cleave nucleotide
strands.
(2) RecA protein allows a single strand to invade a double-stranded DNA.
(3) RuvA and RuvB proteins promote branch migration during homologous
recombination.
(4) RuvC protein is resolvase, a protein that resolves the Holliday structure by
cleavage of the DNA.
(5) DNA ligase repairs nicks or cuts in the DNA generated during recombination.
14
*20. A circular molecule of DNA contains 1 million base pairs. If DNA synthesis at a
replication fork occurs at a rate of 100,000 nucleotides per minute, how long will
theta replication require to completely replicate the molecule, assuming that theta
replication is bidirectional? How long will replication of this circular chromosome
take by rolling-circle replication? Ignore replication of the displaced strand in
rolling-circle replication.
In bidirectional replication there are two replication forks, each proceeding at a
rate of 100,000 nucleotides per minute. So, it would require 5 minutes for the
circular DNA molecule to be replicated by bidirectional replication since each fork
could synthesize 500,000 nucleotides (5 minutes 100,000 nucleotides per minute)
within the time period. Since rolling-circle replication is unidirectional and thus
has only one replication fork, 10 minutes will be required to replicate the entire
circular molecule.
21.
3
5
leading
lagging
*23. What would be the effect on DNA replication of mutations that destroyed each of
the following activities in DNA polymerase I?
(a) 3' 5' exonuclease activity
The 3' 5' exonuclease activity is important for proofreading newly
synthesized DNA. If the activity is nonfunctional, then the fidelity of replication
by DNA polymerase I will decrease, resulting in more misincorporated bases in
the DNA.
(b) 5' 3' exonuclease activity
Loss of the 5' 3' exonuclease activity would result in the RNA primers used
to initiate replication not being removed by DNA polymerase I.
(c) 5' 3' polymerase activity
DNA polymerase I would be unable to synthesize new DNA strands if the 5'
3' polymerase activity was destroyed. RNA primers could be removed by DNA
polymerase I using the 5' 3' exonuclease activity, but could not be replaced
by DNA polymerase I.
CHALLENGE QUESTION
24.
Conditional mutations express their mutant phenotype only under certain conditions
(the restrictive conditions) and express the normal phenotype under other
conditions (the permissive conditions). One type of conditional mutation is a
temperature-sensitive mutation, which expresses the mutant phenotype only at
certain temperatures.
Strains of E. coli have been isolated that contain temperature-sensitive
mutations in the genes encoding different components of the replication machinery.
In each of these strains, the protein produced by the mutated gene is nonfunctional
under the restrictive conditions. These strains are grown under permissive
conditions and then abruptly switched to the restrictive condition. After one round
of replication under the restrictive condition, the DNA from each strain is isolated