E1L6 - DNA replication, Mitosis Flashcards
What explains why DNApol adds dNTPs from 5’ to 3’?
Because the 3’ OH is what nucleophilically attacks the inner most phosphate of the incoming dNTP! So we always have to add dNTPs to the 3’ end of the growing strand. That’s why we always say “5’ to 3’”!
Why is the 5’ to 3’ synthesis important for proofreading ability?
It all boils down to the availability of a triphosphate bond in the dNTP, to add to the growing/errored nucleic acid. If a mismatched dNTP was removed from the 5’ end, it would be irreplaceable since the adjacent dNTP will have no triphosphate bond available to be cleaved, as the correct dNTP is added. On the other hand, if the mismatched dNTP is removed from the 3’ end, the adjacent dNTP’s 3’ OH will be available to attack the uncleaved triphosphate bond of the correct dNTP floating around!
Which strand, leading or lagging, needs more primers in replication?
Lagging strand, because each Okazaki fragment needs its own primer!
Which of the following is NOT one of the 4 characteristics that govern nucleic acid synthesis?
A. Need template DNA strand
B. 5’ to 3’ directionality
C. Polymerase needed to catalyze polymerization
D. Bidirectional polymerization at each replication fork
E. Exonuclease proofreading from 5’ to 3’
E.
I just threw that in there; besides, exonuclease works from 3’ to 5’
Name 6 specialized mechanisms in DNA replication.
- Initiation
- Unwinding
- Priming
- Unidirectional fork movement
- Untangling
- Termination
ORI is more AT rich than CG, why?
AT only have 2 H-bonds, whereas CG have 3 H-bonds. So AT rich is easier to open!
Trick: G and C rhyme with 3!
Which hexameric protein that is an allosteric motor protein that unwinds DNA, and uses ATP in the process because DNA is very stable?
A. Topoisomerase I
B. Topoisomerase II
C. Helicase
D. PCNA “sliding clamp”
C. Helicase
Which of the following is FALSE regarding DNA single-stranded binding proteins?
A. Prevent re-annealing of the template strand to itself
B. Straightens the template strand out, preventing hairpins
C. Cooperative protein binding
D. Binds sugar-phos. backbone; DNA bases are exposed
E. Has two domains - A and B
F. ssBP are needed more in lagging strand, than leading
G. All of the above are TRUE
G. All of those are true.
Which of the following is true of the sliding clamp PCNA?
A. Promotes processive DNA synthesis on leading strand
B. It requires ATP
C. Promotes rapid assembly of replication complex on lagging strand
D. All of the above
D. All of the above are true
Exonuclease activity of DNA pol works in what direction?
“chews back” from the 3’ towards the 5’ end, until the correct Watson-Crick base pairing.
Note: this is opposite from polymerization activity, which is 5’ to 3’ direction!
Endonuclease vs. exonuclease?
Endo - cuts DNA in the middle of a DNA strand
Exo - cuts DNA at the end of strand
Name 2 methods of proofreading we touched on
- 3 to 5 exonuclease activity built in to the polymerase
- Strand-directed mismatch repair
Slide 24
How many bases per turn of DNA? A. 5 B. 6 C. 7.5 D. 10
D. 10
Which of the following is FALSE regarding DNA Topoisomerase I?
A. It relieves supercoiling AHEAD of the replication fork
B. It has a threonine in the active site
C. It doesn’t require ATP
D. It only cleaves 1 of the sugar-phosphate backbones
B.
Tyrosine, not threonine, is in the active site.
Which of the following describe DNA Topoisomerase II?
A. It is a target for cancer therapy
B. It relieves DNA supercoils after replication finishes
C. It nicks BOTH sugar phosphate backbones
D. It requires ATP for the re-annealing processes
E. Inhibiting with Etoposide it may lead to leukemia
F. All of the above
F. All of the above
Etoposide, a Top2 Inhibitor, acts primarily in which phase(s) of the cell cycle?
A. S-phase B. G2 phase C. G1 phase D. M phase E. S-phase and G2 phase
E. S-phase and G2-phase
This makes sense this is when DNA replication is occurring, and finishing up, respectively.
Which of the following is NOT one of the differences between Prokaryotic and Eukaryotic DNA replication?
A. Euk - has multiple origins of replication
B. Euk - mechanisms that prevent redundant replication
C. Euk - DNA replication happens in the nucleus
D. Prok - nucleosomal proteins must be duplicated along with DNA to maintain proper chromosomal organization
D.
This is a property of eukaryotes, not prokaryotes!
Describe an experiment, to help clarify eukaryotic origins or replication.
Radioactive thymidine areas occur in groups of TWO. This tells us that synthesis happens in groups of 2 but doesn’t tell us about the direction. So…
They replaced the 3H-Thymidine with non-radioactive thymidine. Allow cells to replicate more…and so now, during this 2nd window, there is much less radioactivity. Regions replicated later would have less radioactivity - so this tells us the ORDER of replication which tells us about directionality.
What does CDK phosphorylate?
- pre-RC causing it to fall off.
- Starting replication. Blue protein that when phosphorylated, indicates ORI is currently replicating - don’t repeat!
See slide 42
T/F: Euchromatin is replicated before heterochromatin
T. Euchromatin is more active so we need it first!
Which of the following cell lines express telomerase?
A. Germ-line
B. Cancer cells
C. Stem cells in the crypts of colon tissue
D. All of the above
D. All of the above
What prevents a DNA strand from being recognized as a double-stranded break?
Telomeres
Slide 52
What is replicative senescence?
If telomeres are too short, then it won’t replicate. So it’s a fuse - a clock for replication cycles.
Describe a mechanism that explains >28 CAG repeats, resulting in Huntington’s disease.
DNA Replication slippage…slide 56
