E1L6 - DNA replication, Mitosis

Question 1

What explains why DNApol adds dNTPs from 5’ to 3’?

Answer 1

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’”!

Question 2

Why is the 5’ to 3’ synthesis important for proofreading ability?

Answer 2

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!

Question 3

Which strand, leading or lagging, needs more primers in replication?

Answer 3

Lagging strand, because each Okazaki fragment needs its own primer!

Question 4

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’

Answer 4

E.

I just threw that in there; besides, exonuclease works from 3’ to 5’

Question 5

Name 6 specialized mechanisms in DNA replication.

Answer 5

1. Initiation
2. Unwinding
3. Priming
4. Unidirectional fork movement
5. Untangling
6. Termination

Question 6

ORI is more AT rich than CG, why?

Answer 6

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!

Question 7

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”

Answer 7

C. Helicase

Question 8

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

Answer 8

G. All of those are true.

Question 9

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

Answer 9

D. All of the above are true

Question 10

Exonuclease activity of DNA pol works in what direction?

Answer 10

“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!

Question 11

Endonuclease vs. exonuclease?

Answer 11

Endo - cuts DNA in the middle of a DNA strand

Exo - cuts DNA at the end of strand

Question 12

Name 2 methods of proofreading we touched on

Answer 12

1. 3 to 5 exonuclease activity built in to the polymerase
2. Strand-directed mismatch repair

Slide 24

Question 13

How many bases per turn of DNA?
A. 5
B. 6
C. 7.5
D. 10

Answer 13

D. 10

Question 14

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

Answer 14

B.

Tyrosine, not threonine, is in the active site.

Question 15

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

Answer 15

F. All of the above

Question 16

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

Answer 16

E. S-phase and G2-phase

This makes sense this is when DNA replication is occurring, and finishing up, respectively.

Question 17

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

Answer 17

D.

This is a property of eukaryotes, not prokaryotes!

Question 18

Describe an experiment, to help clarify eukaryotic origins or replication.

Answer 18

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.

Question 19

What does CDK phosphorylate?

Answer 19

1. pre-RC causing it to fall off.
2. Starting replication. Blue protein that when phosphorylated, indicates ORI is currently replicating - don’t repeat!

See slide 42

Question 20

T/F: Euchromatin is replicated before heterochromatin

Answer 20

T. Euchromatin is more active so we need it first!

Question 21

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

Answer 21

D. All of the above

Question 22

What prevents a DNA strand from being recognized as a double-stranded break?

Answer 22

Telomeres

Slide 52

Question 23

What is replicative senescence?

Answer 23

If telomeres are too short, then it won’t replicate. So it’s a fuse - a clock for replication cycles.

Question 24

Describe a mechanism that explains >28 CAG repeats, resulting in Huntington’s disease.

Answer 24

DNA Replication slippage…slide 56

Question 25

Diseases of anticipation

Answer 25

Fragile X
Friedrich Ataxia
Huntingtons
Myotonic Dystrophy
Progressive Myoclonus Epilepsy I

The more repeats, the more chance of seeing the disease. Repeats increase over generations, and age of onset decrease over generations.

Penetrance increases with repeat quantity!

Question 26

Challenge question:

Which of the following processes does NOT require the cleavage of the a high energy phosphodiester bond?

A. Proofreading by DNA pol
B. Polymerization by DNA pol
C. Ligation of Okazaki fragments by DNA ligase
D. Topoisomerase I activity
E. Topoisomerase II activity

Answer 26

D. Topoisomerase I (no ATP or dNTP required)

Note: Ligation requires ATP (slide 21)

Question 27

Your data shows that DNA pol is making 1 error every 10,000 base-pairs. You introduce a knockout that eliminates exonuclease activity. How many errors are made now?

A. 1 per 10 BP
B. 1 per 100 BP
C. 1 per 1000 BP
D. 1 per 10,000 BP

Answer 27

C. 1 per 1,000 BP

In class, the prof. said exonuclease activity reduces errors by 100 times. So eliminating the exonuclease activity would increase errors 100-fold.

Question 28

Telomerase is needed when the ____ end of template DNA has no room for _____

A. 3’ , Helicase to bind
B. 3’ , Primase to synthesize a primer
C. 5’ , Okazaki fragments to hybridize
D. 5’ , Primase to synthesize a primer

Answer 28

B. 3’ , Primase to synthesize a primer

Question 29

T/F: telomeres reach an “optimal length” after successive replications, regardless of their length pre-replication.

Answer 29

True

Question 30

Telomeres function to protect single-stranded chromosome ends from:
A. Recombination
B. Fusion
C. Being recognized as damaged DNA
D. Replicative senescence
F. All of the above

Answer 30

F. All of the above