Bacterial replication Flashcards
Which strand is synthesized continuously?
Leading strand
Which strand is synthesized discontinuously and forms Okazaki fragments?
Lagging strand
What is the sequence of the origin of replication (ori)?
sequence with high A:T content where proteins will bind, separate the double stranded DNA, and produce 2 replication forks
How many ori per chromosome?
In prokaryotic: 1
In eukaryotic: hundreds/thousands
Eukaryotic requires synchronization where prokaryotic does not
What 4 things are required to prepare the ori for replication in E. coli?
3 core proteins: DnaA, DnaB (Helicase), DnaC, and 1 accessory protein SSB (Single stranded DNA binding protein)
Function of DnaA
DNA binding domain specific to ori
Multimerization with DNA wrapping relaxes DNA at A:T region
Function of DnaB
Hexameric helicase that unwinds dsDNA into ssDNA
Function of DnaC
assists with loading helicase
Function of SSB
keeps the two ssDNA strands apart and prevents reannealing
How does DnaA protein recognize origin of replication?
- One helix in HTH fits nicely in the major groove
- has some proteins that interact with minor groove to add stability
- part of HTH interacts with phosphate backbone (not sequence specific)
Describe base pair interactions at the consensus sequence (ori)
not many mutations, and is less mutable because function is essential to survival (unless co-evolution between consensus sequence and protein)
describe initiation at the Ori
-specific binding at DnaA sites leads to cooperative coating of approx 20 DnaA proteins to the right half of the origin around DnaA (requires ATP)
- DnaA multimerizes and causes DNA to wrap around it and bind more DNA, while removing twists from DNA and allowing it to be opened by spontaneously dentaturing
How does helicase assemble
- 6 DnaC bound to 6 DnaB allow assembly of single DnaB hexameric ring around each strand
- DnaC is released and helicase will use ATP to move in a 5’ to 3’ direction
- because the DNA is encircled, the enzyme is highly processive meaning it won’t fall off
how does helicase work?
- hexamer works as 3 pairs with each subunit on opposite sides
- each subunit extends a peptide loop which can contact phosphate backbone of coiled ssDNA
- ATP binding and hydrolysis drive each protein through 3 shape conformations (ATP-bound : extended, ADP-bound: middle, Empty: low
- each opposing pair generates some force so when they work in sequence there is continuous progression that pulls DNA apart
What is primase?
5’ to 3’ DNA dependent RNA polymerase that adds fragments on the lagging strand
How does primase work?
- each hexameric ring binds 2/3 molecules of primase
- as helicase travels, primase produces an RNA primer (10-14nt long) every 1500-2000 nt
what is found ahead of bacterial replication fork?
-helicase unwinding causes positive supercoiling ahead of replication fork
-topoisomerase II and gyrase convert positive supercoil into negative supercoil by forming double-strand break and removing two twists
- double strand break occurs 450 000 times in E. coli genome (4.5Mb) because one turn = 10 bp
Pol III classifications
core enzyme: minimum subunits needed to synthesize DNA
holoenzyme: core plus extras
What 3 things make up the Pol III core enzyme structure?
heterotrimer contains subunits
alpha: polymerase with 5’ to 3’ activity
epsilon: proofreader with 3’ to 5’ exonuclease activity
theta: stabilizes epsilon and increases exo rate
what makes up the pol III holoenzyme?
- Pol III core (x2-3: one for each strand + one for Okazaki fragments)
- sliding clamp
- clamp loader
Function of pol III core in holoenzyme
extends the DNA copy from the RNA primer
Structure and function of sliding clamp
dimer that acts as a mobile tether and prevents pol III from releasing target DNA therefore increasing processivity
Structure and function of clamp loader
ATPase that repositions the sliding clamp when initiating a new Okazaki fragment and synchronizes leading and lagging strands
Pol III processivity
-slow synthesis with only 10-15 nucleotides before separation from DNA
- addition of sliding clamp increases is to about 500 000 for the leading strand
- about 500-1000 nucleotides added per second
pol III error rate
- approx. 1 in 10 000 nucleotides will be incroporated incorrectly
- of those, only 1 in 1000 will not get removed by 3’ to 5’ exonuclease
- combined error rate is 1 in 10^7
- about 30/100 correct nucleotides are also removed by 3’ to 5’ exonuclease (false pos.)
rough math on E. coli
- E. coli genome is 4.5 Mb
- single origin gives bidirectional replication fork = 2.25 Mb of synthesis in each direction
-pol III goes 500-1000 nt/sec - replication takes at least 2,250,000/1000 seconds
- 2250 seconds = 37.5 minutes but we know E. coli divides in 20 minutes
How does E. coli replicate it’s genome fast enough for cell division?
“born pregnant” with overlapping cell cycles and multifork replication
- up to 12 replication forks in one cell under optimal conditions
- replication initiates 3 times before one is finished
- allows E. coli to replicate in 20 minutes
does the leading strand require a primer?
yes, it comes from the first primer synthesized when replication begins
What happens to the primer on the lagging strand?
Pol III does not remove or displace the RNA primer on the lagging strand
- RNAse H or DNA pol I can remove the primer
What is RNAse H?
can cleave bond between adjacent ribonucleotides in RNA:DNA hybrids only
What is Pol I?
first polymerase discovered and described in 1956, was noted that there was too much in a cell (hundreds of copies) and it worked too slowly (10nt/sec) to be the primary polymerase
What type of activity does pol I have?
5’ to 3’ pol
5’ to 3’ exo
3’ to 5’ exo (error check)
Pol I function in the lab
used for lagging strand cleanup by nick translation
- can be used in the lab to synthesize labeled DNA
Ligase overall function
- After the primer is removed by RNAse H or Pol I the nicks on the lagging strand must be sealed so that the Okazaki fragments are attached to eachother continuously
- Ligase mediates the reaction using NAD+ or ATP to add AMP to the 3’ end and then swab that bond with the 5’ end phosphate to release AMP
- if the 5’ phosphate has been removed there is no ligation - so dephosphorylated DNA cannot be sealed on its own
what is the solution to seal dephosphorylated DNA
if the 5’ nucleotide is removed, the next nucleotide has a phosphate. DNA pol I can move it one base forward and then it can be sealed by ligase
