7.2 - Chromsome Replication

Question 1

How many origins of replication do bacteria have?

Answer 1

Bacteria have 1 origin of replication (oriC) per circular chromosome

Question 2

How many origins of replication do Archaea (like eukaruotes) have?

Answer 2

Multiple per chromsome

Question 3

How is chromsome replication different to DNA replication?

Answer 3

DNA is packaged inside chromosomes so, to completely copy the whole chromosome, multiple DNA replications must occur inside it simultaneously

Question 4

What steps are needed for chromsome replication?

Answer 4

(1) DnaA-ATP assembly at oriC
(2) Helicase assembly on open DNA
(3) Primase & polymerase + clamp assembly and clamp loader assembly
(4) Rep factory assembly

Question 5

What is DnaA

Answer 5

The key player in initiating chromosome replication.

Question 6

Why is DnaA a good target for antibiotics?

Answer 6

All Eubacteria (i.e. bacteria) have DnaA and all of them use it. Since it is only common to bacteria, it is a good target for antibiotics

Question 7

What are the two forms of DnaA?

Answer 7

(1) The active form: when it is bound to ATP.

(2) The inactive form: when it hydrolyses ATP, and is now bound to ADP

Question 8

If a cell wants to initiate replication what does it need?

Answer 8

A lot of DnaA-ATP. DnaA-ATP initiates replication by binding to and unwinding an AT region in the DNA at oriC, in the first stage of chromosome replication, and recruiting other proteins to the origin

Question 9

What does DnaA-ATP bind to?

Answer 9

Repeated 9-mer sequence within oriC (called DnaA box). One oriC has several repeated 9-mer sequences. It binds as a protein helix that wraps around the DNA helix, resulting in the formation of structures that recruit other proteins.

Question 10

What does the binding of DnaA-ATP lead to?

Answer 10

The separation of strands, using the energy from ATP hydrolysis, at the 13-mer repeat sequence.

Question 11

What is recruited to to the site of replication after DnaA-ATP binding?

Answer 11

2 hexameric DNA helicases (DnaB) and 2 DNA helicase loaders (DnaC)

Question 12

What does DnaC do before it is released?

Answer 12

DnaC will open DnaB ring and place the ring around the ssDNA at the oriC. So each single strand has now one DNAB bound around it. Binding of these hexameric helicases represents the commitment step.

Question 13

What do DNA helicases do (DnaB)?

Answer 13

DNA helicases (DnaB) will use energy from ATP hydrolysis to separate DNA at each replication fork. Each DnaB recruits one DNA primase called DnaG that synthesizes an RNA primer on the strand to which DnaB is bound.

Question 14

What is used to extend DNA to the right?

Answer 14

The top RNA primer (on the 3’ -> 5’ DNA strand)

Question 15

What is used to extend DNA to the left?

Answer 15

RNA primer in the bottom (on the 5’ -> 3’ DNA strand)

Question 16

What is recruited to the DNA template near the first RNA primers?

Answer 16

A complex made of: 2 DNA polymerase III, one clamp loader and one open sliding clamp (DnaN)

Question 17

How is DnaN loaded onto the DNA template near the RNA primer?

Answer 17

With the help of the clamp loader.

Question 18

What does the sliding clamp/DnaN do?

Answer 18

The sliding clamp is a protein that serves as a processivity-promoting factor in bacterial DNA replication. It will bind DNA pol III and prevent it from dissociating from the DNA template. DNA sliding clamp also known as \beta subunit of Pol III holoenzyme is encoded by DnaN genes.

Question 19

What happens once the DNA sliding clamp is properly loaded?

Answer 19

DNA pol III will bind to it and start DNA replication from the 3’ hydroxyl end of the RNA primer, after DNA gyrase unwinds the DNA upstream.

Question 20

What happens as DNA helicase opens up the replication fork?

Answer 20

The leading strand is continuously replicated whereas the lagging strand is replicated in fragments called Okazaki fragments

Question 21

What are single stranded bringing proteins? (SSBPs)

Answer 21

As one Okazaki fragment is being synthesized, the un-replicated lagging strand further down in the replisome will be covered with SSBs. SSBs will protect all ssDNA during the process of DNA replication

Question 22

How does replication on the lagging strand work?

Answer 22

As the lagging-strand DNA polymerase synthesizes one Okazaki fragment, that part of the lagging strand is being looped out (i.e. is forming a loop). When the lagging-strand polymerase bumps into the 5’ end of a previously synthesized Okazaki fragment, the DNA polymerase is released and the DNA clamp is disengaged.

Question 23

What happens after DNA helicase has moved approximately 1000 bases?

Answer 23

Another RNA primer will be synthesized on the lagging strand, the clamp loader will load another clamp to which DNA polymerase will associate and form a new Okazaki fragment. The lagging strand has several Okazaki fragments.

Question 24

What are the RNA primers of Okazaki fragments cleaved by?

Answer 24

RNaseH, then DNA pol I uses the 3’ OH group of the Okazaki fragments to fill in the gaps left by RNA primers with DNA nucleotides. DNA Pol III can also remove RNA primers itself but it is less efficient

Question 25

How does the DNA molecule become continuous as the final step?

Answer 25

DNA ligase seals all the gaps on the lagging strand, resulting in a continuous DNA molecule. DNA ligase in E. coli uses energy from NAD, not ATP.

Question 26

What are ter sites?

Answer 26

While DNA replication occurs, a protein binds helicase and causes replication to pause at sites termed “ter sites”, which are basically pause sites for replication forks.

Question 27

Why are ter sites useful?

Answer 27

If the DNA pol III from one replication fork has fallen off, pausing provides the time for another pol III to be recruited before replication is resumed.

Question 28

What is the signal for replication to be initiated?

Answer 28

When DnaA levels are high

Question 29

What do rep protein aggregate into?

Answer 29

Foci

Question 30

How were were able to determine that rep proteins aggregate into foci?

Answer 30

Fusing PolC to GFP allowed to determine that Rep proteins aggregate into foci.

Question 31

Why are Topoisomerases II (gyrase) present at the end of replication forks?

Answer 31

To release the tension created by the helicases

Question 32

E.coli can divide every 20 min, but it takes 100 mins to replicate its chromosome from one oriC. How does it do this?

Answer 32

By starting a 2nd rep cycle before the 1st one has ended

Question 33

What is an example of checkpoint control?

Answer 33

E coli have nulcoeoid associated protein (NAP) called SlmA which inhibits FtsZ so if it tries to nucleate and contacts this protein because nucleoid is in the way then SlmA will depolymerize FtsZ. Hence, if nucleus is blocking path then there is no FtsZ formation and not attempted cell division

Question 34

What are homologs?

Answer 34

A gene related to a second gene by descent from a common ancestral DNA sequence. These genes encode proteins that perform similar functions.

Question 35

What are orthologs?

Answer 35

Genes in different species that evolved from a common ancestral gene by speciation. Normally orthologs retain the same function in the course of evolution.

Examples include DnaA/DnaB and orc

Question 36

What are paralogs?

Answer 36

Genes related by duplication within the genome of a given species. Paralogs have evolved different functions even if they are related to the same gene.

Gyrase and Topo IV are paralogs

Question 37

How many times does chromosome replication start in stalked cell types?

Answer 37

There is one chromsome in swarmer cells and when they change into stalk cells they initiate replication only once. In swarmer cell the protein CtrA binds origin and represses it