DNA Replication and Transfer

Question 1

Define replicon

Answer 1

Unit of genome where DNA is replicated = each containing an origin for replication initiation

Question 2

Define origin

Answer 2

Sequence of DNA where replication is initiated

Question 3

Define plasmid

Answer 3

Autonomous circular DNA that makes up a separate replicon

Question 4

Define single-copy replication

Answer 4

Replicates once every time bacterial chromosome replicates

Question 5

Define multi-copy replication

Answer 5

Greater number of copies per individual bacterial cell

Question 6

Describe the replication fork

Answer 6

Replication fork = initiated at origin and moves along DNA

Bidirection

Question 7

When is replication bidirectional?

Answer 7

When 1 origin creates 2 replication forks that move in opposite directions

Question 8

Describe a bacterial genome

Answer 8

Usually a single circular replicon
Replicate bidirectionally from single origin

Question 9

How do replica chromosomes split into daughter cells?

Answer 9

Decatenated

Question 10

What does oriC contain?

Answer 10

11 palendromic GATC reapeats
Methylated on ADENINE on both strands

Question 11

What enzyme methylates the adenine in palindromic oriC?

Answer 11

Dam methylase

Question 12

What type of DNA is produced from replication?

Answer 12

Hemimethylated DNA = cannot initiate replication

Question 13

What does the origin need to initiate replication?

Answer 13

Only fully methylated origins can initiate replication

Question 14

How long before new origin is remethylated?

Answer 14

13 minute delay until methylated by Dam methylase

Question 15

What is required for delaying rereplication?

Answer 15

SeqA = binds to hemimethylated DNA
Prevents origin being remethylated

Question 16

For initiation to occur what is needed?

Answer 16

There must be membrane or cell wall synthesis

Question 17

What is the effect of penicillin?

Answer 17

Inhibits cell wall synthesis = blocking replication initiation

Question 18

What is the bacteria licensing factor?

Answer 18

DnaA = inactivated or destroyed after one round of replication

Question 19

What does DnaA need to perform its role?

Answer 19

ATP

Question 20

What is the role of DnaA-ATP?

Answer 20

Melts DNA by binding short repeated sequence that is fully methylated

Question 21

What is the role of DnaB?

Answer 21

ATP hydrolysis-dependent 5’to3’ helicase

Question 22

What is the role of DnaC?

Answer 22

Chaperone = represses the helicase activity of DnaB

Question 23

What is the DnaB/C complex called and where does it bind?

Answer 23

Prepriming complex that binds origin

Helicase is inactive until DnaC leaves

Question 24

What is the role of DnaG?

Answer 24

Primase = binds to helicase complex

Question 25

What does a primase do?

Answer 25

Synthesizes an RNA chain Providing priming end for DNA replication

Question 26

What forms the replication fork?

Answer 26

Hexamer of DnaB = helicase

Question 27

What is gyrase and where does it bind?

Answer 27

Type II topoisomerase Binds ahead of replication fork = relieves strain placed in the double helix as it unravels

Question 28

How does DNAp carry out proofreading?

Answer 28

3' to 5' exonuclease that excises incorrectly paired bases

Question 29

What is processitivity?

Answer 29

Tendancy to remain on a single template rather than dissociate and re-associate

Question 30

What has a 5' to 3' exonuclease?

Answer 30

DNAp I

Question 31

What does DNAp need to initiate DNA synthesis?

Answer 31

RNA primer = synthesized by primase

Question 32

What sub-complexes does DNAp holoenzyme have?

Answer 32

Each monomeric unit has Catalytic core, dimerization subunit and processivity component

Question 33

What is one catalytic core associated with?

Answer 33

One core = associated with one template strand

Question 34

What is the role of the clamp loader?

Answer 34

Places processivity subunit on DNA These two subunits for a beta clamp around DNA

Question 35

What is the role of dimerizing subunit t?

Answer 35

Links the two catalytic cores together

Question 36

What happens to the clamp on the leading and lagging strand?

Answer 36

Leading strans is processive because its clamp keeps it on DNA Clamp on lagging strand dissociates at end of each Okazaki fragment and reassembles for next fragment

Question 37

How are each Okazaki fragments initiated?

Answer 37

Helicase DnaB is responsible for interacting with primase DnaG to initiate

Question 38

What is the role of DNAp I?

Answer 38

Removes RNA primer = using 5' to 3' exonuclease Replaces it with DNA

Question 39

What happens when a replication fork reaches damaged DNA and how can bacteria avoid death?

Answer 39

Replication fork stalls and may collapse Lesion bypass or homologous recombination

Question 40

How does lesion bypass work?

Answer 40

Error-prone DNAp IV = replicates past the lesion allowing replication to continue This may produce mutations in the sequence

Question 41

What are the 3 gene transfer processes in prok?

Answer 41

Transformation = naked DNA Conjugation = bacteria-mediated Transduction = phage-mediated

Question 42

What is an epsiome?

Answer 42

Segment of DNA = can exist and replicate either autonomously in the cytoplasm or as part of chromosome

Question 43

What are two methods of gene transfer via transformation?

Answer 43

CaCl2 then heatshock Electroporation

Question 44

What is conjugation?

Answer 44

Process where genetic material is transferred by F plasmid F = fertility factor

Question 45

What is the free F factor?

Answer 45

Replicon maintained at the level of 1 plasmid It can be integrated into bacterial chromosome

Question 46

What are tra genes and where are they located?

Answer 46

Encoding transfer function = pilus synthesis and assembly, cell pairing nicking at oriT Located in an operon

Question 47

What is the mechanism of conjugation?

Answer 47

Rolling circle mechanism Nick at origin by TraY/I TraY/I unwinds DNA Elongation of growing strand displaces old strand After one revolution displaced strand reaches unit length TraI bound single strand enters recipient

Question 48

What strains are made when F integrates into bacterial chromosome?

Answer 48

High-frequency of recombination strains Plasmid must recombine with bacterial chromosome to become a Hfr cell

Question 49

What happens in Hfr x F- mating?

Answer 49

F- cells almost never acquire F+ phenotype Because only the first part of F is transferred

Question 50

What is an exconjugant?

Answer 50

Each of the cells that separates after conjugation has taken place

Question 51

What are prototrophs vs auzotrophs?

Answer 51

Prototrophs Wild-type strain = has minimal requirement for nutrient supplements Auxotrophs Mutant strain = lost ability to synthesize nutrients (amino acids)