B: DNA replication

Question 1

What is the difference between Go, G1, G2, S and M phases of the cell cycle?

Answer 1

G1 = cell grows and prepares for cell division, checks cell cycle checkpoints - if they are not approved cell stops growing.
G0 = non-dividing state
S = DNA replication phase
G2 = Cell prepares for mitosis by growing, increasing organelles, producing proteins.
M = Mitosis + cell division

Question 2

What is an origin of replication?

Answer 2

Point on DNA where replication begins.

Question 3

What is the difference between NTPs and dNTPs?

Answer 3

NTPs have ribose as sugar, dNTPs have deoxyribose

Question 4

List the items required by DNA polymerase for DNA synthesis

Answer 4

• dNTPS
• Primer
• DNA phosphate
• Template

Question 5

Difference between DNA and RNA polymerase?

Answer 5

DNA polymerase produces double stranded DNA molecule, RNA polymerase produces single stranded molecule.

Question 6

What is a primer?

Answer 6

short existing piece of DNA/RNA that allows DNA polymerase to make new DNA

Question 7

What is the direction of DNA synthesis?

Answer 7

5’ to 3’

Question 8

What is the basic DNA polymerase error rate?

Answer 8

1x10-5 = in vitro (not compatible with human life)
1x10-10 = in vivo (actual number)

Question 9

Describe proof reading by DNA polymerase

Answer 9

Carried out by 3’-5’ exonuclease in DNA polymerase, digests DNA starting at 3’ and moving towards 5’.
A misincorporated nucleotide will slow down polymerisation and increase activity of exonuclease.
3’-5’ exonuclease excises misincorporated nucleotide and DNA polymerase then resynthesises excised section and continues on.

Question 10

Describe mismatch repair

Answer 10

Sometimes a misincorporated nucleotide is not excised during proofreading and creates a mismatched DNA pair.
This is recognised by DNA mismatch repair system and corrected, mismatch repair system must be able to distinguish between template + new strand of DNA to remove mismatched base.

Question 11

Explain the differences between DNA polymerase proof reading and mismatch repair

Answer 11

Proofreading corrects errors during DNA replication, mismatch repair corrects errors after replication.

Question 12

What is HNPCC and what are the characteristics of HNPCC + genes that cause it

Answer 12

Hereditary Nonpolyposis colorectal cancer
Amsterdam criteria:
- 3 relatives over 2 generations with colorectal cancer
- 2 must be first degree relatives
- 1 must be under 50 at time of diagnosis
- FAP (familial adenomotous polyposis) must be excluded

Mutations in MMR genes that cause HNPCC:
- MLH1
- MSH2
- MSH3
- MSH6
- PMS1
- PMS2

Question 13

Explain how DNA replication is initiated

Answer 13

• Recognition: Origin Recognition Complex (ORC proteins) bind to origin of replication
• Melting: Proteins are ori recruit additional proteins and melt the DNA
• Unwinding: double helix needs to be unwound -carried out by helicases to form a replication bubble at ori. Single stranded binding protein binds to DNA to keep it single stranded so various enzymes can work on it.
• Recruitment: ori will recruit proteins that replicate the DNA
• Primer synthesis: RNA primer is synthesised at ori by DNA primase. Supercoiling needs to be relieved by topoisomerase. Once primer is synthesised, DNA polymerase can start DNA synthesis in 5’-3’ direction.

Eytan:
- Recognition: ORC in eukaryotes, DnaA in prokaryotes recgognize and bind the ori
- Melting: of DNA at ori by additional recruited proteins
- Unwinding: helicases form a replication bubble at the ori to make room for initiation proteins to access the DNA at the ori
- Recruitment: proteins at ori recruit proteins that will replicate DNA (including DNA polymerase)
- Primer synthesis: DNA primase synthesizes RNA primer at ori; supercoiling relieved by topoisomerases 1 and 2 in human, DNA gyrase in prokaryotes
- DNA synthesis: by DNA polymerase in a 5’-3’ direction

Question 14

Chemotherapeutic agent etoposide inhibits:

Answer 14

DNA topoisomerase

Question 15

Silent mutation

Answer 15

Change of codon, but still codes for same amino acid –> usually no effect

Question 16

Missense mutation

Answer 16

Change of codon, codes for different amino acid –> may have major, minor or no effect

Question 17

Nonsense mutation

Answer 17

Change of codon, codes for STOP codon –> usually major effect (kills function of protein)

Question 18

Fen-1 and RNAseHI

Answer 18

5’-3’ exonuclease removes RNA primer

Question 19

Etoposide, anthracyclines, mitoxantrone?

Answer 19

inhibit topoisomerase II and cause cell death –> anti-cancer agents

Question 20

Fluoroquinolones + aminocoumarin?

Answer 20

inhibit DNA gyrase + kill bacteria –> antibiotics

Question 21

What does a telomere do?

Answer 21

Extends 3’ end by TTAGGG

Question 22

Enzymes in E.coli and human responsible for DNA unwinding

Answer 22

DNA helicase in both

Question 23

Enzymes in E.coli and human responsible for primer synthesis

Answer 23

DNA primase in both

Question 24

Enzymes in E.coli and human responsible for main DNA synthesis

Answer 24

E. coli = DNA polymerase 3
Human = DNA polymerase alpha/epsilon

Question 25

Enzymes in E.coli and human responsible for RNA primer removal

Answer 25

E. coli = DNA polymerase 1 (5'-3' exonuclease) Human = Fen1 and RNAseH1 (5'-3' exonuclease)

Question 26

Enzymes in E.coli and human responsible for relieving coiling

Answer 26

E. coli = DNA gyrase Human = Topoisomerase II

Question 27

Enzymes in E.coli and human responsible for binding DNA together

Answer 27

DNA ligase in both

Question 28

Typical start codon?

Answer 28

AUG

Question 29

Stop codons?

Answer 29

UGA, UAG, UAA

Question 30

How does DNA replication terminate in E.Coli

Answer 30

Genome is circular - replication fork proceeds around circle to a termination site opposite ori. Contains 10 ter sequences, tus protein interacts with ter sequences to initiate termination. Completed replication genomes are intertwined and relieved by topoisomerase IV.

Question 31

How DNA replication terminate in eukaryotes

Answer 31

Telomere