DNA Replication

Question 1

What is a replicon?

Answer 1

a unit of the genome in which DNA is replicated; each replicon contains an origin for initiation of replication

Question 2

What is an origin?

Answer 2

a sequence of DNA at which replication is initiated

Question 3

What is a plasmid?

Answer 3

an autonomous circular DNA that constitutes a separate replicon

Question 4

How is replication bidirectional?

Answer 4

an origin creates 2 replication forks that move in opposite directions

Question 5

What is the origin of E. coli?

Answer 5

oriC

Question 6

What do ter sites on the bacterial genome do?

Answer 6

cause termination if the replication forks go too far

Question 7

What regulates initiation of bacterial replication?

Answer 7

methylation of the bacterial origin

Question 8

What does oriC contain?

Answer 8

11 palindromic GATC repeats that are methylated on adenine on both strands by Dam methylase

Question 9

What does seqA do?

Answer 9

bind to hemi-methylated DNA and prevent the origin from being remethylated

Question 10

What is the licensing factor of bacterial DNA replication?

Answer 10

dnaA

Question 11

What is licensing factor?

Answer 11

a factor necessary for replication that is inactivated or destroyed after one round of replication

Question 12

What does DnaA-ATP do?

Answer 12

bind to the fully methylated oriC sequences and form an oligomeric complex that melts DNA

Question 13

What is DnaB?

Answer 13

an ATP hydrolysis-dependent 5’ to 3’ helicase

Question 14

What is DnaC?

Answer 14

a chaperone that represses the helicase activity of DnaB

Question 15

What is DnaG?

Answer 15

a primase that releases DnaC allowing DnaB helicase to become active and create the replication fork

Question 16

What does a primase do?

Answer 16

synthesise an RNA chain that provides the priming end for DNA replication

Question 17

What forms the replication fork?

Answer 17

• hexamer of DnaB
• gyrase (type II topoisomerase)
• SSBs

Question 18

What do topoisomerases do?

Answer 18

bind to the double helix ahead of the replication fork and relieve the strain placed on the double helix as it unravels

Question 19

What does DNA polymerase do?

Answer 19

add nucleotides to the 3’ OH end of the growing chain so that it grows in the 5’ to 3’ direction

Question 20

What are the 5 types of DNA polymerase and what are their functions?

Answer 20

• I = major repair enzyme
• II = replication restart
• III = replicase
• IV = translesion replication
• V = translesion replication

Question 21

What do DNA polymerases control?

Answer 21

the fidelity of replication

Question 22

What is proofreading?

Answer 22

a mechanism for correcting errors in DNA synthesis that involves scrutiny of individual units after they have been added to the chain

Question 23

What is processivity?

Answer 23

the tendency of a polymerase to remain bound to DNA in a single template rather than to dissociate and re-associate

Question 24

How can DNA polymerases excise incorrectly paired bases?

Answer 24

by their 3’ to 5’ exonuclease activity

Question 25

What does DNA polymerase I have?

Answer 25

5’ to 3’ exonuclease activity

Question 26

How is fidelity of replication increased?

Answer 26

by proofreading (~100 to ~1000x more)

Question 27

What is semi-discontinuous replication?

Answer 27

the mode of replication in which one new strand is synthesised continuously (leading) while the other is synthesised discontinuously (lagging)

Question 28

What do all DNA polymerases require to initiate DNA synthesis?

Answer 28

a 3’OH priming end

Question 29

How can a priming end be produced?

Answer 29

- RNA primer - nick in DNA - priming protein

Question 30

What is the E. coli replicase?

Answer 30

DNA polymerase III - 900kD complex with a dimeric structure

Question 31

What does each subunit of DNA polymerase III have?

Answer 31

- catalytic core - dimerisation subunit - processivity component

Question 32

What does a clamp loader do?

Answer 32

place the processivity subunits on DNA, where they form a circular β clamp around DNA

Question 33

How is the catalytic core of the DNA polymerase on the leading strand processive?

Answer 33

its clamp keeps it on the DNA

Question 34

What links the 2 catalytic cores of DNA polymerase together?

Answer 34

2 copies of the dimering subunit tau

Question 35

What does the clamp associated with the catalytic core of the DNA polymerase on the lagging strand do?

Answer 35

dissociate at the end of each Okazaki fragment and reassemble for the next fragment

Question 36

What initiates each Okazaki fragment?

Answer 36

helicase DnaB interacting with the primase DnaG

Question 37

What links Okazaki fragments together?

Answer 37

DNA ligase

Question 38

What does DNA polymerase I in E. coli do?

Answer 38

use its unique 5' to 3' exonuclease activity to remove the RNA primer while simultaneously replacing it with a DNA sequence extended from the 3’ OH end of the next fragment

Question 39

What do eukaryotic DNA polymerase delta and epsilon do respectively?

Answer 39

- delta = elongates lagging strand - epsilon = elongates leading strand

Question 40

How can bacteria avoid death when a replication fork stalls at damaged DNA?

Answer 40

- lesion bypass - recombination

Question 41

What do E. coli DNA polymerase IV and V do in the case of DNA lesions?

Answer 41

incorporate a non-complimentary base into the daughter strand

Question 42

What does lesion bypass require?

Answer 42

error-prone DNA polymerase temporary replacement with DNA polymerase III of the replisome

Question 43

What is an episome?

Answer 43

a segment of DNA that can exist and replicate either autonomously in the cytoplasm or as part of a chromosome, mainly found in bacteria

Question 44

What is transformation?

Answer 44

one mode of horizontal gene transfer in bacteria, where a bacterium takes up a piece of DNA floating in its environment

Question 45

How can transformation be carried out in vitro?

Answer 45

CaCl2 heat shock and electroporation

Question 46

What is conjugation?

Answer 46

the process in which two bacteria come in contact and transfer genetic material mediated by F plasmid

Question 47

What is a free F plasmid?

Answer 47

a replicon that is maintained at the level of 1 plasmid/bacterial chromosome and can be integrated into the bacterial chromosome

Question 48

What happens to the F- recipient after conjugation?

Answer 48

it is converted to F+ (except in Hrf transfer)

Question 49

Where are Tra genes encoding transfer functions found?

Answer 49

in the operon

Question 50

Give examples of Tra gene transfer functions

Answer 50

- pilus synthesis and assembly - cell pairing - nicking at oriT

Question 51

When are Hfr strains formed?

Answer 51

when F integrates into bacterial chromosomes

Question 52

Why do most F- cells not acquire an F+ phenotype in Hfr x F- mating?

Answer 52

only the first part of F is transferred

Question 53

What is the order of genes transferred in Hfr x F- mating?

Answer 53

azi-ton-lac-gal

Question 54

Which genes have the highest frequency of being transferred?

Answer 54

those nearest the oriT

Question 55

Which genes are more frequently represented in recombinants?

Answer 55

those that are transferred early

Question 56

What are prototrophs?

Answer 56

wild-type strain that have minimal requirement for nutrient supplements

Question 57

What are auxotrophs?

Answer 57

mutant strain that have lost the ability to synthesise a nutrient e.g. amino acids

Question 58

How can recombinants be selected exclusively?

Answer 58

- counter selection against parental strains using antibiotic e.g. streptomycin - selection using antibiotics or ability to utilise a sugar e.g. lactose

Question 59

Why can't lac- grow in the mammal medium with only lactose?

Answer 59

it cannot utilise lactose

Question 60

How is an F' recombinant formed?

Answer 60

by the improper excision of F from the bacterial chromosome

Question 61

What are the 2 types of transduction?

Answer 61

- generalised - specialised

Question 62

When does generalised transduction take place?

Answer 62

during the lytic cycle

Question 63

What happens in generalised transduction?

Answer 63

randomly sized fragments are packed into the phage and homologous recombination may occur in the recipient bacteria

Question 64

What is specialised transduction?

Answer 64

transfer of specific portions of the bacterial genome carried out by temperate phages that have integrated their DNA into the host chromosome

Question 65

When does specialised transduction take place?

Answer 65

when the lysogen is induced to go into lytic phase (phage induction)

Question 66

What does integration and excision of phage lambda involve?

Answer 66

site-specific recombination