DLW04 - DNA Replication and DNA Transfer

Question 1

Define the term “replicon”

Answer 1

A replicon is a unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication.

Question 2

Define the term “plasmid”

Answer 2

A plasmid is an autonomous circular DNA that constitues a separate replicon.

Question 3

What is meant by plasmid single copy replication?

Answer 3

Replication of the plasmid that takes place every time the bacterial chromosome replicates.

Question 4

What is meant by plasmid multicopy replication?

Answer 4

The plasmid is present in a greater number of copies per individual bacterial cell.

Question 5

Briefly describe the Messelson-Stahl experiment.

Answer 5

The experiment demonstrates semiconservative replication. Replication is accomplished by separation of the strands of a parental duplex, with each strand then acting as a template for the synthesis of a complementary strand.
In short, the newly synthesised DNA will have one parental strand and one daughter strand.

Question 6

Which experiment demonstrates the semiconservative nature of DNA replication?

Answer 6

The Messelson-Stahl experiment.

Question 7

Describe the bacterial genome in relation to DNA replication.

Answer 7

The bacterial genome is a single circular replicon. In E. coli, the origin of replication is oriC (about 245 bp in length). The bacterial genome also contains ter sites cause termination.
Replicated chromosomes may be catenated upon completion of replication.

Question 8

OriC contains eleven palindromic repeats that are methylated on adenine as part of replication initiation. State the sequence of these repeats.

Answer 8

GATC

Question 9

Fully methylated DNA is unable to initiate replication as it prevents DNA polymerase III binding. TRUE or FALSE?

Answer 9

FALSE. Full methylation is required for the initiation of replication.

Question 10

What enzyme catalyses the methylation of DNA for replication initiation? Which base is methylated?

Answer 10

Dam methylase; adenine.

Question 11

What does “Dam” stand for?

Answer 11

Deoxyadenosine methylase

Question 12

Describe how DNA replication is delayed.

Answer 12

Only fully methylated origins can initiate replication. Dam methylase is responsible for methylating the origin of replication at adenine of the GATC repeats. However, after a single round of replicated, hemi-methylated DNA is generated. SeqA then binds to hemi-methylated DNA to prevent remethylation of the origin.

Question 13

What is the function of SeqA?

Answer 13

SeqA binds to hemi-methylated DNA, preventing the origin from being methylated. This results in the delay of DNA replication.

Question 14

If Penicillin is added, DNA replication is not halted. However, as the cell continues dividing, the lack of cell wall synthesis results in the lysis of the bacterial cell. TRUE or FALSE?

Answer 14

FALSE. Without cell wall synthesis, DNA replication will not even initiate.

Question 15

State the function of DnaA

Answer 15

DnaA is the licensing factor. DnaA-ATP binds to fully methylated short repeated sequences and forms an oligomeric complex that melts DNA.

Question 16

State the function of DnaB

Answer 16

DnaB is an ATP-dependent 5’ to 3’ helicase. DnaB also interacts with DnaG to initiate each Okazaki fragment.

Question 17

State the function of DnaC

Answer 17

Six DnaC monomers bind a DnaB hexamer, repressing the helicase activity of DnaB.

Question 18

State the function of DnaG

Answer 18

DnaG is a primase and also serves to release DnaC, allowing DnaB to become active. DnaG is also responsible for interacting with DnaB to initiate each Okazaki fragment.

Question 19

State the function of gyrase (topoisomerase)

Answer 19

Topoisomerase binds to the double helix ahead of the replication fork and relieves the strain placed on the double helix as it unravels.

Question 20

State the function of single-stranded binding proteins

Answer 20

SSBPs bind to the newly separated strands, preventing them from re-annealing.

Question 21

The new DNA strand is synthesised in the 5’ to 3’ direction. TRUE or FALSE?

Answer 21

TRUE.

Question 22

Define the term “proofreading”

Answer 22

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

Question 23

Define the term “processivity”

Answer 23

Processivity refers to an enzymes ability to catalyse consecutive reactions without releasing its substrate.

Question 24

State the direction of exonuclease activity for DNA polymerase I.

Answer 24

5’ to 3’

Question 25

State the direction of exonuclease activity for DNA polymerase III.

Answer 25

3’ to 5’

Question 26

Describe semi-discontinuous replication.

Answer 26

The mode of replication in which one new strand is synthesised continuously while the other is synthesised discontinuously.

Question 27

All DNA polymerases require a 3’ -OH priming end to initiate DNA synthesis. TRUE or FALSE?

Answer 27

TRUE.

Question 28

List the three ways a priming end can be provided.

Answer 28

1. Nick in DNA
2. Priming protein, which provides a priming nucleotide.
3. RNA primer synthesised by a primase.

Question 29

Describe the structure of DNA polymerase III in E. coli.

Answer 29

DNA polymerase III is a 900 kD complex with a dimeric structure. Each monomeric unit has a catalytic core, dimerisation subunit, and a processivity component.

Question 30

Briefly describe how DNA polymerase III in E. coli is loaded onto DNA.

Answer 30

DNA polymerase III has a dimeric structure. Each catalytic core is associated with its own template strand.
A clamp loader places the processivity subunits on DNA, where they form a circular beta clamp around DNA.
The dimerisation subunits link the two catalytic cores together

Question 31

Explain processivity, using DNA replication as an example.

Answer 31

The catalytic core of DNA polymerase on the leading strand is processive because its clamp keeps it on the DNA.
On the other hand, the clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment.

Question 32

Describe how Okazaki fragments are combined to form a continuous strand of DNA.

Answer 32

1. Each Okazaki fragment starts with a primer and stops before the next fragment.
2. In E. coli, DNA polymerase I removes the primer (5’ to 3’ exonuclease) and replaces it with DNA using the nick as a primer.
3. Ligase seals the nick.

Question 33

State the difference in priming between E. coli and eukaryotes in terms of enzymes involved.

Answer 33

E. coli requires DnaG for priming, while eukaryotes utilise either DNA polymerase alpha or primase.

Question 34

State the difference in DNA replication catalysis between E. coli and eukaryotes in terms of enzymes involved.

Answer 34

E. coli requires two DNA polymerase III catalytic cores.
Eukaryotes, however, require two different polymerases: DNA polymerase delta synthesises the lagging strand while DNA polymerase epsilon synthesises the leading strand.

Question 35

DNA damage can cause the replication fork to stall and collapse. Bacterial cells have two possible ways to avoid death due to replication failing. State and explain the two ways.

Answer 35

1. Lesion bypass. Replication by an error-prone DNA polymerase (pol IV and pol V), incorporating a non-complementary base into the daughter strand. DNA pol III has to be quickly swapped back after the bypass has finished.
2. Homologous recombination

Question 36

State three ways in which prokaryotic gene transfer can occur.

Answer 36

1. Transformation
2. Conjugation
3. Transduction

Question 37

Define the term “episome”

Answer 37

An episome is 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 38

State the two ways bacterial transformation can be carried out

Answer 38

1. CaCl2 with heat shock

2. Electroporation

Question 39

Define conjugation

Answer 39

Conjugation is a process in which two bacteria come in contact and transfer genetic material.

Question 40

How many F plasmids are present in a single cell?

Answer 40

One. It is a single copy replicon.

Question 41

The F- recipient will always be converted into F+ after conjugation. TRUE or FALSE?

Answer 41

FALSE. For high frequency of recombination (Hfr) strains, the recipient cell will usually not gain the entire F plasmid, hence will remain as F-.

Question 42

Briefly state the genes encoded for by the F plasmid.

Answer 42

The F plasmid is approx. 94.5 kb and carries tra genes encoding transfer functions (pilus synthesis and assembly, cell pairing, nicking at oriT). The F plasmid may also carry other beneficial genes (such as antibiotics resistance)

Question 43

The F plasmid is transferred via the rolling circle mechanism. Briefly describe the rolling circle mechanism.

Answer 43

1. The F plasmid is nicked on one strand.
2. The nicked strand is then transferred, 5’ end first, to the recipient cell.
3. Meanwhile, elongation is carried out at the 3’ end, displacing the transferred strand.
4. The transferred strand recircularises in the recipient cell and the complementary strand is synthesised soon after.

Question 44

How do Hfr strains form?

Answer 44

Hfr strains form when the F plasmid integrates into bacterial chromosome.

Question 45

Why do F- cells almost never acquire an F+ phenotype in Hfr and F- mating?

Answer 45

The F plasmid is integrated into the bacterial chromosome in Hfr transfer. The bacterial genome is large, and conjugation is often unstable. Conjugation will terminate before the entire F factor can be transferred. Only the first part of F is transferred.

Question 46

What is meant by temporary partial diploidy?

Answer 46

When a recipient bacterial cell receives an extraneous DNA fragment, it may possess two copies of the same gene (diploidy). Since the DNA fragments are often times not the entire bacterial chromosome, only some genes will have two copies (partial). The DNA fragments will be quickly destroyed or undergo homologous recombination (temporary).

Question 47

Briefly describe how chromosomal mapping would work with conjugation.

Answer 47

1. The order of genes transferred can be determined by conjugation.
2. Genes near the oriT have the highest frequency of being transferred.
3. Genes transferred early are more frequently represented in the recombinants than those late.

Question 48

Define the term F’

Answer 48

F’ (F prime) is formed by the improper excision of F from the bacterial chromosome. The F’ plasmid can carry as much as 15% of the genome, thus providing partial diploidy when transferred into a recipient strain.

Question 49

Briefly describe the two types of transduction.

Answer 49

1. Generalised transduction: any part of the bacterial genome can be transferred. Occurs during the lytic cycle.
2. Specialised (restricted tranduction): transfer of only specific portions of the bacterial genome. Carried out by temperate phages that have integrated their DNA into the host chromosome.

Question 50

The amount of DNA transferred during generalised transduction is constant regardless of the type of phage involved. TRUE or FALSE?

Answer 50

FALSE. Bacteriophage 22 carries ~50 E. coli genes while bacteriophage P1 carries ~100 E. coli genes.

Question 51

Briefly describe specialised trasduction.

Answer 51

Temperate phages can form lysogens by integrating themselves into the host genome.
Phage particles thus carry both phage DNA and flanking bacterial DNA. Only bacterial DNA adjacent to the prophage insertion site is packaged.
This occurs only when the lysogen enters the lytic phase.