Lecture 5 - DNA Replication

Question 1

DNA double helix consists of:

Answer 1

sugar-phosphate backbone with paired bases hydrogen bonded together

Question 2

DNA double helix can assemble from its component strands:

Answer 2

• unzipped by breaking hydrogen bonds between the bases
• this allows new strands to be synthesised by matching up bases to the old strands
• this process of DNA replication allows DNA to be copied when cells divide

Question 3

how is DNA synthesis catalysed?

Answer 3

DNA synthesis is catalysed by enzymes known DNA polymerases and they occur in the 5’-3’ direction

Question 4

DNA replication is semi-conservative:

Answer 4

because a newly made DNA molecule therefore has one daughter strand and one parent strand

Question 5

what are the three possible principles of DNA replication:

Answer 5

(a) semi-conservative → accepted model
(b) dispersive
(c) conservative

Question 6

meselson-stahl experiment:

Answer 6

bacterium were grown in a light medium and a heavy medium and their centrifuged DNA showed two separate band locations - however when these bands were transferred to their heavier/lighter counterpart their newly centrifuged values were in between - indicating semi-conservative/dispersive replication

Question 7

how does the new strand form in DNA synthesis?

Answer 7

The dNTP base pairs with the template strand through H bonding

DNA polymerase catalyses formation of a new phospho-diester bond between the 3’OH of the preceding nucleotide in the chain with the new base paired nucleotide

Question 8

RNA primes DNA synthesis:

Answer 8

because DNA polymerases cannot initiate a new DNA chain on their own, each new DNA strand must be initiated by a synthesis of a short RNA (primer) that is extended by the DNA polymerase

the RNA primer part of the chain is then removed

Question 9

why always 5’-3’?

Answer 9

DNA synthesis always goes in a 5’-3’ direction

its likely always in this direction because proofreading is chemically difficult if synthesis occurred in the 3’-5’ direction

Question 10

higher eukaryotic DNA polymerases:

Answer 10

eukaryotic cells express a range of DNA polymerases with slightly different characteristics

some are focused of bulk DNA replication at the replication fork whilst others are focused on the bypass of damaged DNA / repair pathways

Question 11

rate of encorporation of incorrect dNTPs=

Answer 11

10,000 slower

Question 12

rate of encorporation of rNTPs=

Answer 12

1000 fold slower

Question 13

what prevents DNA polymerase from rNTP binding?

Answer 13

“discriminator” amino acids

Question 14

how does the DNA polymerase interact with the RNA primer?

Answer 14

DNA polymerase grips the primer template junction like a hand

Question 15

what is the final error rate during DNA replication?

Answer 15

the final error rate is 1 mistake per every 1010 nucleotides added

Question 16

what do DNA polymerases used in DNA replication have next to the active site?

Answer 16

next to the active site DNA polymerases have an exonuclease site

Question 17

what is the purpose of exonuclease activity?

Answer 17

incorrectly incorporated bases can be caught by the exonuclease activity, allowing the DNA polymerase to have another go at incorporating the correct base

Question 18

because DNA polymerase can only synthesise 20-100 base pairs before releasing the template, how does it stay in place and not fall off?

Answer 18

•Sliding clamp encircles newly synthesised dsDNA and holds DNA pol

• DNA pol prevented from diffusing away from primer: template junction

Question 19

the replisome:

Answer 19

DNA replication requires a mix of DNA unwinding, DNA polymerase and DNA stabilization factors to come together in a highly processive molecular machine – the replisome

Question 20

“trombone slide” model of replication:

Answer 20

• the leading strand can be synthesised continuously in a 5’-3’ direction
• the lagging strand is looped out so that it passed through the polymerase active site in a 3’-5’ direction, allowing synthesis to occur in the 5’-3’ direction

Question 21

how are RNA primer fragments removed from the DNA molecule?

Answer 21

RNA fragments are removed via the RNAase enzyme for DNA polymerase to fill the gap for DNA ligaments to make the final seal

Question 22

enzymes for DNA replication:

Answer 22

•Primase
•DNA polymerases (2)
•Clamps
•Single-stranded DNA-binding protein, to keep strands apart
•DNA ligase
•Helicase, to unwind DNA
•Topoisomerase, to relax DNA

Question 23

what is different about the origins of replication in eukaryotes and prokaryotes?

Answer 23

bacteria have one single origin per chromosome whilst eukaryotic chromosomes have many origins to compensate for larger genomes and slower speed

Question 24

how do RNA primers differ in eukaryotes and prokaryotes?

Answer 24

RNA primers are longer in eukaryotes and shorter in prokaryotes

Question 25

how do okazaki fragments differ in prokaryotes and eukaryotes?

Answer 25

okazaki fragments are longer in prokaryotes than they are in eukaryotes

Question 26

what is the function of PCR (polymerase chain reaction)?

Answer 26

‘amplifies’ (i.e. selectively makes lots of) specific DNA sequences

Question 27

what does PCR require?

Answer 27

PCR requires: DNA polymerase, oligonucleotide ‘primers’

Question 28

each cycle of PCR requires 3 steps:

Answer 28

(1) separating DNA strands (95C) (2) ‘annealing’ of primers. (40-60C) (3) DNA synthesis (70C)

Question 29

because the polymerase chain reaction has stages that exceed to 95C what is crucial that we do?

Answer 29

use thermal protected enzymes such taq DNA polymerase which comes from thermophilic organisms used to these temperatures

Question 30

PCR summary:

Answer 30

• Development of the PCR has revolutionised molecular biology and has widespread applications • Tiny amounts of DNA can be amplified in a few hours • PCR works because DNA polymerases require primers

Question 31

where does a replisome assemble?

Answer 31

a replisome assembles at each replication fork with a complex set of enzymes

Question 32

orientation of DNA replication:

Answer 32

DNA replication is bi-directional