Flashcards in Module 2 part 2 Deck (32):
Explain the general function of E. coli pol 1 and its exonuclease presence.
DNA repair and Okazaki fragment processing (same as the DNA pol 1 we just went over)
pol 1 contains both 5'-3' and 3'-5' exonuclease activity
Explain the general function of E. coli pol 2 and its exonuclease presence.
not fully understood pol
involved in translesion synthesis
it ONLY contains 3'-5' exonuclease
Explain the general function of E. coli pol 3 and its exonuclease presence.
pol 3 is responsible for chromosome replication. It is aka replicase
it ONLY contains 3'-5' exonuclease
Explain the general function of E. coli pol 4 and 5 and their exonuclease presence.
they have NO EXONUCLEASE ACTIVITY
If there is a damaged site of the DNA the replication fork may be stuck
pol 4/5 can place incorrect nucleotides into the the growing strand to get the DNA replication fork to start moving again.
this causes mutation, but a stuck replication fork can be life or death without these pol's.
This is translesion synthesis
how many sites of replication does E. coli have? what are they called?
only one and its called oriC
oriC is 250bp long
What are the two known features of OriC? explain each
1) DnaA 9-mer sites: 4 copies of a 9 nucleotide sequence repeat. These 4 sites are where the bacterial initiator protein dnaA binds too.
2) A=T rich 13-mer repeats: 3 direct repeats (DR = same direction repeats) of 13 base pairs. This is the DNA unwinding element which readily unwinds upon dnaA binding
What is a replicon?
All of the generated DNA (new strand) from one origin. In E. Coli the entire chromosome is the replicon
5 steps of E. coli replication activation: Generating the open complex. start with DnaA
DnaA is a member of the AAA+ family. These proteins have a region which can hydrolyze ATP.
- DnaA binds the OriC region where it oligomerizes and wraps around the DNA which strains it
- With ATP activity, dnaA destabilizes A=T rich 13-mer repeats in oriC. Facilitated by a protein called HU this forms a small ssDNA bubble called the open complex
In generating the open complex in the activation of E. coli replication, what proteins are required and what do they form?
- AAA+ dnaA protein
- Binds OriC, and destabilizes A=T rich 13-mer repeats
- HU protein
- forms a dnaA-ATP-OriC-HU complex that forms an open complex bubble
5 steps of E. coli replication activation: Activation of replication origin
start with dnaC
- two dnaB (helicase) molecules attach to each DNA strand
- This is achieved by dnaC which loads on each dnaB
- Once they are on the protein-DNA complex is called the pre priming complex
what is dnaC called? how does it get dnaB onto the ssDNA?
it is a AAA+ protein which is called Helicase loading protein
- It pries open dnaB hexameric ring and slips it around the ssDNA
- theATP bound form of dnaC binds to dnaB which represses the helicase activity
- when the ATP is hydrolyzed the dnaC is ejected off dnaB
5 steps of E. coli replication activation: replication fork assembly
start with DnaB
- DnaB goes on to bind ATP
- this ATP enables dnaB translocation which kicks off DnaA
- ATP bound also allows further unwinding of the DNA
- this unwinding generates a positive supercoil stress which must be removed by Topoisomerase ( it repositions DNA to remove stress)
- the newly unwound DNA is coated with single stranded binding proteins SSB to protect it
- Primase forms primers at each fork
What is the beta clamp?
a ring shaped homodimer that is apart of pol 3 which moves ahead of the pol 3 core and increases processivity. When this is active, pol 3 is a haloenzyme
5 steps of E. coli replication activation: leading strand
- now the primer is formed, it directs attachment of the beta clamp on the leading strand. pol 3 is now active on the leading strand
5 steps of E. coli replication activation: lagging strand
continued dnaB (helicase) activity allows for lagging strand priming and Okazaki fragment synthesis
List the steps in order of the initiation of replication in E. Coli (5)
1) dnaA oligomerizes upon binding the OriC region. It wraps DNA to form an open complex. this is aided by HU
2) DnaC loads a dnaB onto both strands = pre priming complex
3) The ATP on dnaB allows it to translocate and dislodge the dnaA protein. unwinding generates positive supercoil stress which is removed by topoisomerase. Primase makes primers
4) primers direct B clamp loading and assembly of pol 3 haloenzyme complex.
5) continued dnaB helicase action allows lagging strand synthesis
What stops the cell from replicating the genome again to make 4 daughter cells at the end of the cell cycle
inactivation of oriC
What is methylation?
a form of inhibiting DNA replication. In the OriC region there is 11 Dam methylase sites that contain the base pair pattern GATC.
SeqA is a protein that hides these sites and prevents dnaB binding. Dam methylase works in between seqA dissociation - reassociation cycles to methylate these sites and allow dnaB to bind
What is dnaA - ATP - hydrolysis
When dnaA is kicked off during the replication fork synthesis it hydrolyses its ATP to ADP. dnaA - ADP can rebind to DNA but it cannot generate an open complex therefore inhibiting further replication
What is hda clamp binding?
hda protein binds to the beta clamp and this stimulates dnaA to hydrolyze its ATP. it is a catalyst
What is inhibiting tension from RNA polymerase mean?
4th mechanism to regulate replication. When there is supercoil strain RNA polymerase can prompt replication. therefore we use rifa protein to inhibit RNA pol.
Explain the steps involved in assembly of the replication fork at oriC (4)
1) ATP allows translocation of dnaB which dislodges dnaA
2) dnaB moves unwinding the DNA. topoisomerase reduces positive supercoil stress
3) Newly unwound DNA is coated with single stranded binding proteins for protection
4) RNA primers are then synthesized by primase
how many times per cycle do eukaryotic origins fire (activate)?
once per origin per cell cycle
What stage of the cell cycle does replication and chromosome separation occur?
replication = S
separation = M
Explain the initiation of replication in S. Cerevasa.
this eukaryotic cell has conserved replication regions called ARS which are A=T rich.
1) ORC binds to DNA and is faciliated by CdC6 (similar to HU)
2) This complex loads on MCM2-7 (like B)
3) This loading is facilitated by Cdt1 (like C)
4) this forms the pre-replication complex
In E. coli, what makes up the replisome
- Pol 3 haloenzyme
- DnaB helicase
how is dnaB connected to the pol 3 core?
dnaB connects to the core through the clamp loaders tau subunits within the pol 3 enzyme (look at diagram)
how many tau subunits are there?
true or false, each pol 3 core has a beta clamp attached to it. this complex is then conncectec to the clamp loader through tau subunits
Explain the beta clamp structure and function
- it is a homo dimer that is a circle protein.
- it converts pol 3 from a distributive protein to a processive protein by holding the pol 3 onto the DNA
explain pol 3 core structure and function
same structure as pol 1 (fingers, thumb, and palm)
it is where replication occurs.