Bentzen 3 - DNA Replication

Question 1

What are the three proposed methods of DNA replication? Which one is right?

Answer 1

Conservative replication

Dispersive replication

• Semiconservative replication -

Question 2

Describe the Meselson and Stahl experiment. What is EDGC?

Answer 2

1. Grew E. coli on 15N medium for many generations
2. Switched some cells to 14N medium
3. Used equilibrium density gradient centrifugation (EDGC) to determine isotope composition of DNA of the three samples (15N, 1st round of 14N and 2nd round of 14N)

Question 3

How were the conservative and dispersive models of DNA replication proved wrong? What was their predictions?

Answer 3

Conservative predicted 50% old and 50% new.

Dispersive predicted 100% intermediate (mix of old and new)

The results were 100% intermediate after the bacteria was switched to the light medium and then 50/50 once allowed to replicate again. Which is what the semiconservative model predicted.

Question 4

What is semi-conservative replication?

Answer 4

Two DNA strands of parental molecule seperate

Each parental strand serves as a template for replication.

Rules of base pairing ensure genetic info is correctly copied

Each daughter DNA molecule consists of one parental strand and one newly synthesized strand

Question 5

What are the four minimum requirements for DNA synthesis?

Answer 5

1. Template of single stranded DNA (ssDNA)
2. All four deoxyribonucleoside 5’ triphosphates (dNTPs)
3. DNA polymerase and other enzymes and proteins
4. Free 3’-OH group

Question 6

What are the two mechanisms involved in DNA polymerization at the nulceoside level (by DNA polymerase)

Answer 6

Catalysis of covalently bonding the phosphate group to a ribose in the backbone (forming phosphodiester bonds) and base pairing.

Question 7

What units does DNA polymerase attach to the growing chain?

Answer 7

deoxyribonucleoside 5’ triphosphates (dNTPs)

Question 8

What does DNA polymerase catalyze?

Answer 8

Phosphodiester bonds

Question 9

What is meant by DNA polymerase being unable to make a DNA chain de novo?

Answer 9

It can’t make a new chain, only extend a preexisting chain

Question 10

What direction does DNA polymerase elongate the chain in?

Answer 10

5’ to 3’ direction

Question 11

What direction is the template strand read in by DNA polymerase?

Answer 11

3’ to 5’

Question 12

What is the origin of replication?

Answer 12

The specific nucleotide sequence where replication begins.

Question 13

Is replication bi-directional or uni-directional?

Answer 13

Bi-directional

Question 14

What is a replicon?

Answer 14

Any DNA molecule or a region of DNA that replicates from a single origin of replication.

Question 15

What is the leading strand?

Answer 15

This is where synthesis of DNA follows the replication fork. There is a leading strand at each fork

Question 16

List two types of DNA replication in circular genomes and one type in linear genomes

Answer 16

Circular

• Theta replication (bacteria, E. coli)
• Rolling circle replication (viruses)

Linear
- Linear Replication (eukaryotes)

Question 17

What is theta replication?

Answer 17

A type of circular DNA replication, where there is a single replicon. Replication is bidirectional at each fork and replication terminates on other side of circular DNA.

Question 18

What is rolling circle DNA replication?

Answer 18

There is no replication bubble and there is uncoupling of the replication of the two strands of the DNA molecule.

Replication begins at the 3’ OH and ends with cleavage at the 5’ end of the leaving strand of DNA. Can be thought of as unwinding string from a spool.

Question 19

What proteins are involved in initiation of prokaryotic DNA replication? (3). What do they do?

Answer 19

Initiator proteins (bind to the origin of replication, oriC, and unwind a short section of DNA)

Helicase (and other proteins bind to ssDNA, bind to the lagging strand template and break hydrogen bonds)

Single stranded binding proteins (keep DNA strands separated).

Question 20

What proteins are involved in unwinding of DNA during DNA replication in prokaryotes? (2) What do they do?

Answer 20

DNA Helicase, seperates the two DNA strands be breaking hydrogen bonds

DNA gyrase (a topoisomerase) travels ahead of the replication fork and alleviates supercoiling caused by unwinding

Question 21

How does DNA gyrase work?

Answer 21

It travels ahead of the replication fork and alleviates supercoiling caused by unwinding, by breaking and then resealing strands to relieve stress.

Question 22

What proteins are envolved in chain elongation? What do they do?

Answer 22

Primase adds an RNA primer sequence at the origin, it provides a free 3’ OH for the DNA polymerase to use. RNA is used because the production of it does not need a free 3’ end.

DNA Polymerase (III is the principle, I removes and replaces RNA primers with DNA)

DNA ligase seals the nick in the sugar phosphate backbone`

Question 23

How many DNA polymerases are there in E coli?

What does DNA polymerase I do?
What does DNA polymerase III do?

Answer 23

5

They all have 5’ to 3’ polymerase activity

Polymerase I has exonuclease activity where it removes and replaces RNA primers with DNA in the 5’ to 3’ direction, it also has polymerase activity where it fills in the gaps (also in 5’ to 3’ direction)

DNA polymerase III is the principle replication enzyme.

Question 24

What is the error rate of DNA polymerase without proof reading? With proof reading?

What is the mechanism for proof reading?

Answer 24

1 per 100,000 nucleotides without proof reading and 1 per 10 million with proof reading.

Polymerase III is stalled when it comes across incorrect base pairing. It then uses 3’ to 5’ exonuclease activity to remove the incorrect nucleotide.

Question 25

How many replication origins are activated in eukaryotic DNA replication? What is each cluster known as?

Answer 25

20-80. Each cluster of replication origins is known as a replication unit.

Question 26

How do eukaryotic cells do controlled initiation in DNA replication? What protein shuts this down?

Answer 26

1. An origin must be selected or ‘licenses’ (replication licensing factor binds)
2. Origin activation (replication begins)
3. Once activated/replicated an origin is deactivated using Geminin

Question 27

What is the speed of replication in euakryotes and prokaryotes? How long does it take each? Why?

Answer 27

E. coli takes about 40 minutes at 1000 bp/s with one origin. Humans take 8 hours with 50 bases/sec with over 10000 origins.

Humans can replicate DNA so much more accurately (while at the same time staying efficient) than E. coli by having many origins

Question 28

What is the end replication problem with telomeres?

Answer 28

Telomeres are the ends of linear chromosomes that are made up of G-rich short repeated sequences. They stabilize the chromosome.

Each round of replication leaves up to 200 base pairs of DNA unreplicated at the 3’ end. It is a gap left by the removal of the primer (since there is no 3’ hydroxyl group at the very and, no more DNA can be added).

Question 29

What is the solution to the end replication problem at telomeres?

Answer 29

Telomerase

It is a specialized reverse transcriptase. Entends the end of the parental DNA by RNA-templated DNA synthesis.