-
What are the four phases of the cell cycle? Which
three phases are parts of interphase? What occurs in the cell in each of
the four phases? Rank the four phases in order of length of time spent for
a typical cultured mammalian cell? What is G0 phase?
- Mitoticphase, G1, S (DNA sythesis), G2
- Interphase is G1, S (DNA sythesis), G2
- G1:8-10, S: 6-8 G2:4-6
- G0 phase: cells arrest at G1, waiting for signals to enter S
-
Understand the Meselson and Stahl experiment
shown in Fig. 19-2. What is the conclusion from the experiment? How would
the results be different if the conservative model is correct instead?
-
How does a bacterial chromosome replicate and how
do the two resulting copies separate into two cells?
 - Yes,
-
If eukaryotic DNA replications occur at a much
slower rate (fewer nucleotide pairs/min) than bacterial DNA replications
and eukaryotic genomes are larger, what do eukaryotic cells do to allow
their chromosomes to be duplicated in a reasonable time frame? (Hint: what
are origins of replication and replicons?)
- many replication origins
- A replicon is a replication
- unit.
- One ORI for each replicon.
-
What are the functions of initiator proteins?
Bind to the origin of replication of DNA and initiates unwinding of DNA double helix.
-
Know the mechanism of DNA replication. Include
template, semiconservative nature, replication origin, replication bubble,
replication fork, direction of synthesis, continuous leading strand
synthesis, discontinuous lagging strand synthesis, and Okazaki fragments.
–Template
–Semiconservative
–Replication origins
- •Thousands in human genome
- •Particular sequence that attracts
- initiator proteins
- •Easy to separate two strands,
- thus A-T rich
- –Bidirectional replication forks
- –Rapid
- •Antiparallel parent DNA ® asymmetrical
- replication fork
–Leading strand: continuous synthesis
- –Lagging strand: discontinuous synthesis
- •Okazaki fragments
-
What is the advantage for
growing/extending/polymerizing DNA in the 5’ to 3’ direction as opposed to
the 3’ to 5’ direction?
It allows 3' to 5' proofreading by exonuclease.
-
What are the two catalytic activities of DNA
polymerase III in bacteria (one is 5’ to 3’, the other is 3’ to 5’)?
- •In bacteria, DNA polymerase III is the main
- enzyme for DNA replication. It
- –Adds deoxynucleotides to the 3’ end (5’ to 3’
- polymerization) by forming phosphodiester bonds
- –Hydrolyzes phosphoanhydride bond in PPi to release free energy for
- polymerization
- –Proofreads (3’ to 5’ exonuclease activity) reducing error rate to
- one error in every 107 nucleotide pairs it copies
-
The endergonic reaction of linking nucleotides in
phosphoester bonds is coupled to the exergonic hydrolysis of what bonds?
phosphoanhydride bond in PPi
-
Which three DNA polymerases are found in
eukaryotic cells? How do the functions of the polymerases differ?
- •In eukaryotic cells, DNA polymerase α is the
- main enzyme for initiating DNA replication for both leading and lagging
- strands. It adds to the 3’ end of the RNA primers.
- •DNA polymerases δ and ε are
- both thought to be involved in synthesizing leading and lagging strands. They
- also have 3’ to 5’ exonuclease activity for proofreading. DNA polymerases δ also
- replaces the primer with DNA after it is digested away by RNase H.
- •DNA polymerase adds to a base-paired 3’ end.
- •It adds to a RNA primer, about 10 nucleotides
- long.
- •Primase
- synthesizes RNA primers from scratch by linking ribonucleotides together. It has no proofreading activity.
-
Why is a RNA primer needed in DNA replication?
It is used by DNA polymerase III for DNA synthesis.
-
enzyme is responsible for synthesizing the RNA
primer?
Primase
-
Which enzyme is responsible for breaking down the
RNA primer in prokaryotes? How about in eukaryotes?
- In eukaryotes: RNase H
- in pro: DNA polymerase I 5’ to 3’ exonuclease (removes RNA primer)
-
What are the three catalytic activities of DNA
polymerase I in bacteria?
•In bacteria, DNA polymerase I
–3’ to 5’ exonuclease (for proofreading)
–5’ to 3’ exonuclease (removes RNA primer)
- –5’ to 3’ polymerase (replaces RNA
- primer)
-
What are the functions of DNA helicase, DNA
topoisomerase, single-stranded DNA binding proteins, and DNA ligase in DNA
replication?
- DNA helicase-Unwinds DNA from hydrolysis
- DNAtopoisomerase-introduces
- negative supercoils and thereby relaxes
- positive ones
- Single-stranded DNA binding protei ns: keeps DNA unwound
- DNA ligase- links together okazaki fragments
-
Compare DNA polymerase III and primase in the
following: Does it need a base-paired 3’ end to add (deoxy)nucleotides to?
Does it have proofreading activity? Is it used in the leading strand or
lagging strand synthesis or both?
- DNA poly III needs a based-paired 3' (deoxy)nucleotides
- Primase does not.
- DNA poly proofreads the lagging strand
- Primers have no proofreading activity.
-
Why is there an end-replication problem for
linear DNA? How do eukaryotic cells solve this problem (the answer should
include an explanation of what are telomeres and telomerases and the
functions each serves)?
- The last primers are removed by a 5' to 3' exonuclease, but no DNA polymerase can fill the resulting gaps because there is no 3' OH available to which a nucleotide can be added.
- Then each round of replication generates shorter strands.
- •Telomerase
- –An enzyme composed of protein and RNA
- –The RNA portion is complementary to DNA repeat sequence in telomeres
- –Elongates telomeres
- –Active in germ cells, not expressed in many somatic cells, but can become reactivated
- in tumor/cancer cells
- •Telomeres
- –Repetitive nucleotide sequences found at the ends of eukaryotic chromosomes (100-1500
- tandem repeats of TTAGGG in humans)
- –Allow replication of chromosome ends
- –Indicate true ends of chromosomes
-
In which cell types are telomerases active?
germ cells
-
What are the differences in bacterial vs.
eukaryotic DNA replication? (Hint: consider the number of origin of
replication, the type of DNA polymerases and their roles, shape (linear or
circular) of the chromosome).
- Bacteria contains only one origin of replication.
- Thousands in human genome.
- DNA replication is more rapid in humans.
- In bacteria DNA polymerase III is the main enzyme for replication. DNA poly I is used to proofread and remove/replace RNA primer.
- In euks, DNA poly alpha is the main enzyme for replication.DNA poly omega and epsilon is also involved and is also used for proofreading and places RNA primer
- Bacteria has circular chromosome.
-
What are mutations? What are the consequences of
mutations if they occur in germ cells vs. somatic cells?
- •Mutation: change in nucleotide sequence of
- DNA
- –Random
- –Accumulate with age
- –Mutations in germ cells are passed on to future generation of offspring
- –Mutations in somatic cells affect the individual organism (frequently results in cancer
- as nearly all carcinogens are mutagens)
-
What is the error rate of the DNA replication
machine after proofreading from DNA polymerases?
1 mistake per 10^7 nucleotides
-
What is DNA mismatch repair? By how much does it
improve the accuracy of DNA replication? Which strand of the newly
replicated DNA molecule does it repair? In E. coli, how does the cell
differentiate the two strands?
- •DNA mismatch repair improves accuracy to 1 error per 109 nucleotides copied.
- –Repair the newly synthesized strand only
- –Mutations in genes encoding mismatch repair proteins causes inherited predisposition to certain cancers.
- Repairs only the newly synthesized strand
- The new strand is unmethylated for a while.
-
What are the steps involved in correcting DNA
mismatch?
-
Which two types of DNA damage frequently occur
spontaneously? Describe the damages and to which bases does each typically
occur?
- •Spontaneous DNA damage
- –Occurs
- randomly and equally on both strands
- –Includes
- •Depurination: loss
- of purine (A or
- G) base
- •Deamination: C to U is
- most common
-
DNA damage can also be induced by which four
types of environmental agents? Describe the damages each causes.
–Radiation
- •Exposure
- to UV light (induces thymine dimers)
•X-rays
•Radioactivity
- –Base
- analogs mimic
- nitrogenous bases and get incorporated
- –Base-modifying
- agents alter DNA structure
•Alkylating agent
- –E.g.
- addition of methyl group
- –E.g.
- addition of bulky group
- –Intercalating
- agents distort
- DNA structure
-
If DNA damages are uncorrected, what is a likely consequence
for each?
- •Unrepaired
- mutations can lead to
–Substitution
–Deletion
–Insertion
–DNA replication stop
–Silent (same a.a.)
- –Missense
- (different a.a.)
–Nonsense (stop codon)
- –Frameshift
- (insertions/deletions of nucleotides not in multiples of three; alters the
- reading frame)
-
What are substitution, insertion, and deletions? What
are silent, missense, nonsense, and frameshift mutations?
–Silent (same a.a.)
- –Missense
- (different a.a.)
–Nonsense (stop codon)
- –Frameshift
- (insertions/deletions of nucleotides not in multiples of three; alters the
- reading frame)
-
What are the two types of excision repair? What
is the mechanism of excision repair? Which enzymes are involved and what
are their roles?
- –Base excision repair
- corrects single damaged bases
- –Nucleotide excision repair
- corrects bulkier lesions
- –The repair enzymes that recognize
- and remove the damaged region are specialized to the type of damage
- –DNA polymerases and ligases are
- used in repair
•Bacteria: DNA polymerase I
- •Eukaryotic cells: DNA polymerases
- α, δ, and ε
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