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Genes, Genomes and Biomedical Implications > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

What is a replicon?

A

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

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2
Q

What is an origin?

A

a sequence of DNA at which replication is initiated

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3
Q

What is a plasmid?

A

an autonomous circular DNA that constitutes a separate replicon

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4
Q

How is replication bidirectional?

A

an origin creates 2 replication forks that move in opposite directions

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5
Q

What is the origin of E. coli?

A

oriC

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6
Q

What do ter sites on the bacterial genome do?

A

cause termination if the replication forks go too far

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7
Q

What regulates initiation of bacterial replication?

A

methylation of the bacterial origin

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8
Q

What does oriC contain?

A

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

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9
Q

What does seqA do?

A

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

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10
Q

What is the licensing factor of bacterial DNA replication?

A

dnaA

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11
Q

What is licensing factor?

A

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

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12
Q

What does DnaA-ATP do?

A

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

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13
Q

What is DnaB?

A

an ATP hydrolysis-dependent 5’ to 3’ helicase

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14
Q

What is DnaC?

A

a chaperone that represses the helicase activity of DnaB

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15
Q

What is DnaG?

A

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

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16
Q

What does a primase do?

A

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

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17
Q

What forms the replication fork?

A
  • hexamer of DnaB
  • gyrase (type II topoisomerase)
  • SSBs
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18
Q

What do topoisomerases do?

A

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

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19
Q

What does DNA polymerase do?

A

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

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20
Q

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

A
  • I = major repair enzyme
  • II = replication restart
  • III = replicase
  • IV = translesion replication
  • V = translesion replication
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21
Q

What do DNA polymerases control?

A

the fidelity of replication

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22
Q

What is proofreading?

A

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

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23
Q

What is processivity?

A

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

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24
Q

How can DNA polymerases excise incorrectly paired bases?

A

by their 3’ to 5’ exonuclease activity

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25
Q

What does DNA polymerase I have?

A

5’ to 3’ exonuclease activity

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26
Q

How is fidelity of replication increased?

A

by proofreading (~100 to ~1000x more)

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27
Q

What is semi-discontinuous replication?

A

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

28
Q

What do all DNA polymerases require to initiate DNA synthesis?

A

a 3’OH priming end

29
Q

How can a priming end be produced?

A
  • RNA primer
  • nick in DNA
  • priming protein
30
Q

What is the E. coli replicase?

A

DNA polymerase III - 900kD complex with a dimeric structure

31
Q

What does each subunit of DNA polymerase III have?

A
  • catalytic core
  • dimerisation subunit
  • processivity component
32
Q

What does a clamp loader do?

A

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

33
Q

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

A

its clamp keeps it on the DNA

34
Q

What links the 2 catalytic cores of DNA polymerase together?

A

2 copies of the dimering subunit tau

35
Q

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

A

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

36
Q

What initiates each Okazaki fragment?

A

helicase DnaB interacting with the primase DnaG

37
Q

What links Okazaki fragments together?

A

DNA ligase

38
Q

What does DNA polymerase I in E. coli do?

A

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

39
Q

What do eukaryotic DNA polymerase delta and epsilon do respectively?

A
  • delta = elongates lagging strand
  • epsilon = elongates leading strand
40
Q

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

A
  • lesion bypass
  • recombination
41
Q

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

A

incorporate a non-complimentary base into the daughter strand

42
Q

What does lesion bypass require?

A

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

43
Q

What is an episome?

A

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

44
Q

What is transformation?

A

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

45
Q

How can transformation be carried out in vitro?

A

CaCl2 heat shock and electroporation

46
Q

What is conjugation?

A

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

47
Q

What is a free F plasmid?

A

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

48
Q

What happens to the F- recipient after conjugation?

A

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

49
Q

Where are Tra genes encoding transfer functions found?

A

in the operon

50
Q

Give examples of Tra gene transfer functions

A
  • pilus synthesis and assembly
  • cell pairing
  • nicking at oriT
51
Q

When are Hfr strains formed?

A

when F integrates into bacterial chromosomes

52
Q

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

A

only the first part of F is transferred

53
Q

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

A

azi-ton-lac-gal

54
Q

Which genes have the highest frequency of being transferred?

A

those nearest the oriT

55
Q

Which genes are more frequently represented in recombinants?

A

those that are transferred early

56
Q

What are prototrophs?

A

wild-type strain that have minimal requirement for nutrient supplements

57
Q

What are auxotrophs?

A

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

58
Q

How can recombinants be selected exclusively?

A
  • counter selection against parental strains using antibiotic e.g. streptomycin
  • selection using antibiotics or ability to utilise a sugar e.g. lactose
59
Q

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

A

it cannot utilise lactose

60
Q

How is an F’ recombinant formed?

A

by the improper excision of F from the bacterial chromosome

61
Q

What are the 2 types of transduction?

A
  • generalised
  • specialised
62
Q

When does generalised transduction take place?

A

during the lytic cycle

63
Q

What happens in generalised transduction?

A

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

64
Q

What is specialised transduction?

A

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

65
Q

When does specialised transduction take place?

A

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

66
Q

What does integration and excision of phage lambda involve?

A

site-specific recombination

Genes, Genomes and Biomedical Implications (15 decks)

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