7.3 - Plasmids

Question 1

What are some features of plasmids?

Answer 1

• Plasmids replicate autonomously, independently of the host chromosome and use different mechanisms of replication.
• Plasmids have their own origin of replication and they use cellular machinery to replicate their DNA.

Question 2

What happens to plasmids during cell division?

Answer 2

When a cell is dividing, plasmids will segregate or “partition” to each of the two poles of the cell, in order to allow each daughter cell to have a plasmid.

Question 3

What do some plasmids use for their replication?

Answer 3

DnaA

Question 4

What is a feature of natural plasmids?

Answer 4

They can act like independent organisms

Question 5

What is a feature of artificial plasmids?

Answer 5

They can be used as tools for cloning

Question 6

What is the replicon for replication using PCR?

Answer 6

(1) Melting: temperature
(2) Priming: customized DNA primers
(3) Polymerizing: Taq pol, quick-start brands, etc
(4) Regulators: you and the PCR machine (thermocycler)

Question 7

What is the replicon for replication of E.coli chromosomes?

Answer 7

(1) Melting: DnaA-ATP, DnaB (helicase)
(2) Priming: DnaG
(3) Polymerizing: Pol III + DnaN, Pol I
(4) Regulators: many, target DnaA or oriC

Control goal: cell cycle coordination; very sophisticated

Question 8

What is the replicon for replication from CoIE1 plasmids?

Answer 8

(1) Melting: RNA polymerase will melt the DNA at the site of replication initiation.
(2) Priming: RNase H will form primers.
(3) Polymerizing: This primer will be recognized by DNA pol I which will start elongation. The lagging strand uses DNA pol III to replace the different primers with DNA sequences.

The control goal when we talk about plasmid replication is to produce only a certain number of copies depending on the plasmid.

(4) Regulators: The way the plasmid “knows” when to stop replicating is by the accumulation of a specific antisense RNA. When this antisense RNA accumulates, it will inhibit plasmid replication. This is an example of negative feedback thermostat.

Question 9

How does the iteron work?

Answer 9

Plasmid encode at least one protein called rep protein for replication and borrows most of other protein. Rep binds series of target sites in origin which are repeated (interated). They bind as dimer and handcuff or bind two plasmids together. Replication is closed but if you disrupt handcuffing then system is set up for recruiting protein to initiate replication

Key point: all proteins handcuff molecules together and this gives you sensor for concentration of DNA
If the cytoplasm grows and DNA does not replicate then you are more likely to shift towards equilibrium

Question 10

How might you evolve a seconday chromsome? (Since bacteria typically only have one)

Answer 10

A plasmid could evolve into a chromsome

Question 11

What is an example of a bacteria that has two chromosomes?

Answer 11

Vibrio cholerae which is a close relative of E.coli. Chromosome 1 has origin of replication called OriC which uses DnaA to intiate. Second chromosome is like 1 million bps and bacteria cannot function without it. Its origin of replication is completely different looks more like iteron (clearly derived from plasmid)

Question 12

Do the chromsomes in vibrio cholerae replicate together?

Answer 12

No the large chromsome actually replicates first then the smaller one duplicates. The timing is more sophisticated than you would expect and there is lots of choreography and precise movement of DNA.

Question 13

What are the two types of mechanisms for plasmid segregation?

Answer 13

Push mechanism - separates

Pull mechanism - anchors one molecule then pulls the other

Question 14

What is the main concept behind the push and pull mechanism?

Answer 14

A specific DNA sequence on a chromsome or plasmid acts as a centromere. This recruits a specific protein which binds to the sequence. Another protein acts on this bound protein which results in the required motion.

Question 15

What is an example of push type plasmids?

Answer 15

pR1 segregation

Question 16

in pR1 segregation what protein binds to the DNA sequence which acts as the centromere?

Answer 16

ParR

Question 17

What does ParR do?

Answer 17

Leads to the polymerization of several ParM-ATP (so ParM-ATP binds ParR on “centromere”)

Question 18

What happens during the polymerization process in pR1 segregation?

Answer 18

ParM-ATP becomes ParM-ADP. ParM is very similar to actin filaments

Question 19

What happens to the ParM-ADP filament?

Answer 19

The ParM-ADP filament will eventually hit a ParR molecule on another plasmid. This filament of ParM will make sure that the plasmids are away from each other so that they segregate during cellular division. The plasmids are therefore pushed apart towards the cell’s poles by ParM polymerization, such that after cell division both daughter cells have 1 copy of the plasmid

Question 20

What is an experiment conducted on pull type plasmids?

Answer 20

(1) Live cell experiment with agar pad and nutrients. Cell is on pad and has fluroecent labeled ParB
(2) ParB binds the centromere
(3) Fluorescent micrograph taken every minute
(4) See two sports which separate and stay at poles
(5) This is typical pattern

Question 21

What are GapR proteins?

Answer 21

Proteins that help with separation. Without the protein, chromosomes have trouble separating, cells seem to be separating and collapsing repeatedly. It is a helper protein which means a cell can survive without it

Question 22

How do pull systems work?

Answer 22

(1) ParB binds to an anchoring protein called HubP in vibrio cholerae.
(2) The ParB in chromsome 1 specifically binds and holds the pole.
(3) Whilst one ParB is anchored to HubP, another ParB has a high affinity for ParA which also bound to different parts of chromosome. ParB consumes the ParA it interacts with by hydrolyzing ATP then keeps moving on

Question 23

What centromere does ParB bind to?

Answer 23

ParS which is a DNA sequence

Question 24

How does the pull syster differ in cholobacter?

Answer 24

The anchoring protein used is PopZ.

Question 25

What is another function of ParB bound to centromeres

Answer 25

Loading other proteins. There are a class of proteins called SMC proteins and in eukaryotes these are called condensing proteins which form protein rings. These rings form loops around bundles of DNA and help condense chromosome

Question 26

What are the basic rep protein types?

Answer 26

Polymerase, Nuclease and Ligase

Question 27

What rep protein type is Topoisomerase?

Answer 27

Both a nuclease and a ligase

Question 28

What is another important family of proteins?

Answer 28

AAA+ proteins

Question 29

What are AAA+ proteins?

Answer 29

ATPases Associated with many Activities. They bind and hydrolyze ATP and they all aggregate to form rings or helices. We can think of them as doing microscale work

Question 30

Name two AAA+ proteins

Answer 30

DnaB helicase - contains a 6 member ring of these AAA+ proteins and hydrolyze ATP and unwind DNA.
DnaA - has multiple domains. Bulk of protein is a AAA+ domain which is what allows DNA to aggregate with other molecules on the DNA. ATP is used here as a switch – when ATP is loaded it switches DNA to active conformation to initiate chromosome replication

Question 31

How is the marking of origin of replication different between bacteria, eukarya, and archaea?

Answer 31

In bacteria origin has DnA boxes which bind to origin. Archaea and eukarya use a set of proteins called orc (origin recognition complex proteins). Like DNA most of protein in Orc is AAA+ protein. Acentral AAA+ proteins which got modified and evolved and some became DnA and some became Orcs. It chooses where origin of replication is by binding DNA and marking where replication begins

Question 32

How does the use of origin of replication differ between bacteria, eukarya, and archaea?

Answer 32

In bacteria, almost all chromosomes have one origin of replication but eukaryotes have many so density at origin is much higher. In archaea there are few origins per chromosome.

Question 33

How does the timing of when origin or replication begins differ between bacteria, eukarya, and archaea?

Answer 33

When bacteria initiate chromosome replication, DnA is bound and then gets activated by sufficient concentration od DnaA-ATP which recruits DnaB and is the defining event of replication. In eukaryotes the helicase is not recruited, but loaded well before initiation. Helicases called Mcm (mini chromosome maintenance) which is AAA+ family proteins like DnaB. Typically loaded in M phase or G1 phase and waits for signals to come and release it.

Question 34

How does activation of replication differ between bacteria, eukarya, and archaea?

Answer 34

Bacteria origins of replication can be activated rapidly. Can re replicate as often as you like once replication is initiated. In eukaryotes and archaea there is pre loading, with a single “fire”. Only designed to happen once. Result is very precise duplication where you only duplicate the chromosome once. This means bacteria has the potential for most rapid growth.

Question 35

Do eukarya and archaea need exactly placed origins of replication?

Answer 35

Can get by without origins of replication and do just fine. For example egg cell has nucleus and yolk and everything for rapid growth. Do not need to make more proteins can just go. Eukaryotes do not always replicate slowly this is actually very rapid replication. System does not actually need origin of replication it just needs an orc to be bound to some place and then you get a once per cell cycle firing.