1
Q
E.Coli Pol I features
A
- 1 subunit
- Okazaki fragment processing +DNA repair
- 3’-5’ and 5’-3’ exonuclease
- not used in chromosome replication
2
Q
E.Coli Pol II features
A
- 1 subunit
- Translesion synthesis
- 3’-5’ exonuclease
- possibly used for DNA repair
3
Q
E.Coli Pol III features
A
- 3 subunits
- 3’-5’ exonuclease
- used for chromosome replication, sometimes called replicase
4
Q
E.Coli Pol IV/V features
A
- Pol. IV: 1 subunit, Pol. V: 2 subunits
- no proofreading exonucleases
- translesion DNA polymerases
- used when DNA damage halts replication fork, get replication moving again
5
Q
E.Coli replication stages
A
- Initiation of replication
- Elongation by the replisome
- Termination
6
Q
DnaA 9-mer sites
A
4 copies of 9-nucleotide (9-mer)consensus sequence to which bacterial initiator protein DnaA binds
7
Q
A=T rich 13-mer repeats
A
- to one side of the DnaA 9-mer sites are 3 A=T rich direct repeats (13 BP each)
- repeats known as DNA unwinding element, unwind readily upon binding of initiator
8
Q
Steps of oriC activation
A
- Generate open complex
- activate replication origin
- assembly of E.coli replication forks
- replication initiation and leading strand synthesis
- lagging strand synthesis
9
Q
Generation of Open complex
A
- when bound to oriC, DnaA oligomerizes, + wraps DNA around oligomer complex (strains DNA)
- in presence of ATP, DnaA destabilizes A=T rich 13mer repeats of oriC, forms ssDNA bubble with help of HU protein
- complex formed is DnaA-ATP-oriC-HU (open complex)
10
Q
About DnaA
A
- in AAA+ protein family
- AAA+ family has ATPase activity, binds/hydrolyzes ATP structural domain that assist in protein + DNA conformational changes
11
Q
Activation of Replication origin
A
- ssDNA regions are exposed, 2 hexamers of DnaB helicase assemble ( 1 on each strand)
- DnaC used to load DnaB by prying open ring of DnaB and slipping around ssDNA
- ATP bound DnaC binds to DnaB, represses helicase ability
- hydrolysis of ATP ejects DnaC from DnaB
- protein-DNA assembly at the oriC called prepriming complex
12
Q
Assembly of E.coli replication forks
A
- DnaB binds ATP, allows it to translocate+unwind DNA, dislodging DnaA protein
- unwinding occurs outward in both directions, widens replication bubble
- topoisomerase enzyme relieves supercoil stress
- newly unwound DNA in bubble coated with SSB proteins
- primase cannot interact with DnaB until bubble is 100-200 BP in size
13
Q
Replication initiation and leading strand synthesis
A
-RNA primer directs loading of beta clamp and assembly of leading strand Pol III holoenzyme
14
Q
Beta clamp
A
- component of E.coli Pol. III holoenzyme
- ring shaped homodimer encircles and slides along duplex DNA ahead of Pol III core (attached to Pol III)
- enhances processivity
15
Q
Holoenzyme
A
- active form of an enzyme
- enzyme that requires co-factor but is not bound to it is called apoenzyme
16
Q
Lagging strand synthesis in E.coli
A
- Pol III holoenzyme extends its RNA primer until both Pol III complexes connect w DnaB helicase travelling in same direction on other side of bubble
- priming is followed by clamp loading and synthesis of lagging strand by 2nd Pol III enzyme in the complex
- this generates multiple Okazaki fragments discontinuously
- replication continues until end of template of until another replication fork from an adjacent oriC is reached
17
Q
Initiation in Euk vs. E.coli
A
- Euk have higher DNA content, slower replication forks
- Euk need multiple origins of replications
- Euk origins fire once per cell cycle
18
Q
S. cerevisiae replication (eukaryotic)
A
- origins called ARS (autonomously repeating sequences), AT rich
- indicator called origin recognition complex (ORC), subunits similar to DnaA, ATP required for ORC binding
- after ORC binds to DNA, Cdc6 (AAA+) binds to ORC (similar to HU)
- ORC-Cdc6 complex loads Mcm2-7 complex onto DNA
- Mcm2-7 (DnaB-Like) is circular hexamer, binds to 1 mlc of Cdt1 (DnaC-like)
- this is required before ORC-Cdc6 complex can load Mcm2-7 complex onto DNA
- ABOVE EVENTS OCCUR ONLY IN G1 PHASE
- resulting complex called prereplication complex (preRC)
- after addition of proteins+checkpoints, RC begins to replicate DNA in S phase
19
Q
E.coli replisome
A
- Pol. III holoenzyme, DnaB helicase, primase
- helicase connects to Pol. III core through tau subunits of clamp loader
- connection between DnaB helicase and Pol. III holoenzyme increases speed of DnaB
- 3 tau subunits of tau complex clamp loader bind 3 Pol III cores (same tau subunits that bind DnaB)
- leading strand Pol.III-B-Clamp complex moves continuously w DnaB
- Lagging strand Pol. III-B-clamp complex repeatedly moves on and off DNA to extend multiple RNA primers made by primase
20
Q
B-clamps facilitate processivity
A
- B-clamps ensure contact between Pol III core and DNA
- B-slidiing clamps assembled onto both DNA strands by one clamp loader
21
Q
Clamp loader mechanism
A
-in Pol. III holoenzyme is tau complex
-uses energy of ATP binding to open B-clamp
(can’t bind clamp w/o ATP)
22
Q
About Trombone Model
A
- lagging strand polymerase extends 3’term of okazaki fragment in opposite direction to fork movement
- pol. still part of replisome, moves with fork
- results in DNA loop for each okazaki fragment
- when lagging strand pol. finishes Okazaki fragment, it dissociates from DNA to transfer to a new RNA primer (detaches from B-clamp, associates w new B-clamp at next primer)
23
Q
Steps of Clamp recycling
A
- Pol. III core dissociates from B-clamp following synthesis of Okazaki fragment
- B-clamp site attracts Pol. I, removes RNA primer w 5’-3’ exonuclease - Pol. I dissociates from DNA, leaves ssDNA break
- B-clamp attracts DNA ligase, forms phosphodiester bond to seal break - unoccupied B-clamp opened + unleaded by excess delta subunits of clamp loader
24
Q
Termination of E.coli replication
A
- region halfway around circular chromosome from oriC contains 2 clusters of 23 BP sequences (Ter sites, oriented in opposite directions)
- monomeric Tus protein binds to Ter site, blocks advance of replication fork by stopping DnaB helicase
- Tus-Ter complex is directional, replication forks only blocked when approaching complex from nonpermissive direction
25
Competition between transcription + replication in E.coli chromosome
- replicating bacteria is still transcribing RNA from promoters
- collisions between RNA pol and replication forks happens
- codirectional collisions do not impede fork
- head on collisions cause fork to pause/stall
- most collisions codirectional
26
Steps of topoisomerase mechanism
- type II topoisomerase in prokary is heterodimer, 2 ATPase domains, 2 cleavage core domains
- pulls one piece of catenated (tangled) dsDNA, cleaves with ATP (at cleavage site using Tyr residue)
- Tyr gives up proton to form nucleophile, attacks phosphate in phosphodiester bond
- temporarily covalently links DNA to protein
- once daughter chromosomes separated, hydrolysis rxn reforms phosphodiester bond
27
Problem with eukaryotic termination
- at end of chromosome, once leading strand completely extended, RNA primer at the extreme end needs to be replaced with DNA
- PROBLEM: no 3' term. for the Pol. to extend from, so genetic info in the gap would be lost in next round of replication
- progressive shortening after multiple rounds of replication
28
Solution to eukaryotic termination problem
- ends of euk chromosomes called telomeres (repeats of a unique sequence)
- telomerase reverse transcriptase (TERT) carries non-coding telomerase RNA (TR)
- TR contains 1.5 telomerase repeats, used as template to extend 3' term. of telomere beyond what was replicated
- TERT-TR holoenzyme is called telomerase
- RXN OCCURS IN S PHASE
29
Telomerase process steps
1. @ 3'-term, 3 nucleotides of telomere anneal to 3 RNA nucleotides on telomerase
2. Telomerase extends 3' end of ssDNA by length of one telomere repeat
- telomere carries own template, synthesizes ssDNA
3. after adding telomere repeat, telomerase separates from DNA-RNA hybrid
- repositions on telomere for next extension, acts processively
4. telomerase extended 3' ssDNA term. converted to dsDNA by same priming/polymerization machinery used in chromosome replication
