Module 02 - Section 03 Flashcards
DNA replication in the Model Organism E.coli
Which DNA Polymerases have 3’-5’ exonuclease?
DNA Pol I, II, & III
Which DNA polymerase has 5’-3’ exonuclease?
DNA Pol I
What are the functions of DNA Pol I?
Okazaki fragments processing and DNA repair
What is the function of DNA Pol II?
Translesion synthesis; DNA repair
What is the function of DNA Pol III?
chromosome replication
What is the function of DNA Pol IV
Translesion synthesis; enables replication fork to move past a damaged site
What is the function of DNA Pol V
Translesion synthesis; enables replication fork to move past a damaged site
Which DNA polymerases do not have 3’-5’ exonuclease?
DNA Pol IV and V
Describe the DNA sequence of the origin of replication of E.coli
(1) DNA 9-mer sites: 4x of a 9-nucleotide consensus sequence (DnaA binds to it)
(2) A=T rich 13-mer repeats (3 direct repeats)= unwinding element (unwinds easily upon binding of initiator)
What is a replicon?
All of the DNA replicated from a particular origin of replication
Describe how the Open Complex is generated in E.Coli
(1) DnaA (initiator, AAA+ protein) binds DNA 9-mer sites
(2) DnaA oligomerizes and wraps the DNA around itself like a scarf
(3) DnaA, in the presence of ATP destabilizes A=T rich 13-mer repeats forming a ssDNA bubble - facilitated by HU
What is HU
A small basic histone-like protein that facilitates the formation of the ssDNA bubble
What are AAA+ proteins ?
A family of proteins with ATPase activity that share a common structural domain called the AAA domain, which assist in protein and DNA conformational changes
What is the initiator protein in E.Coli?
DnaA
Describe the activation of the Replication Origin
(1) DnaC pries open the hexameric ring of DnaB and slips it onto the ssDNA at the bubble
(2) DnaC binds tightly to DnaB when ATP-bound, once ATP is hydrolyzed DnaC is ejected
(3) 1 DnaB is assembled on each ssDNA (2 total)
Describe the assembly of the E.Coli replication Forks
(1) DnaB binds ATP, which allows it to translocate and unwind DNA, dislodging the DnaA
(2) Unwinding happens outwards in both direction
(3) Topoisomerase removes the supercoil stress due to unwinding of DNA
(4) newly unwound DNA is coated with single-stranded binding proteins (SSB) to protect it
Describe the replication initiation and leading strand synthesis
(1) RNA is made which directs the loading of the beta clamp and assembly of the Pol III holoenzyme
Describe the components of the Eukaryotics Initiation (4)
(1) highly conserve A sequence (initiator binding)
(2) B1
(3) B2
(4) B3 (A-T rich)
What is the eukaryotic initiator
Origin Recognition complex, which has subunits similar to DnaA and ATP is required for binding ORC to the origin
Describe how the prereplication complex is created in eukaryotes
(1) Cdc6 (also an AAA+ protein) binds to ORC (like HU)
(2) ORC-Cdc6 then loads the Mcm2-7 (DnaB-like) complex onto DNA
(3) Cdt1 (DnaC-like) binds to Mcm2-7 is required for ORC-Cdc6 complex to laod Mcm2-7 onto the DNA
* *Occurs only in G1 phase of the cell cycle
Describe the structure of Beta Clamps
Homodimers shaped like a ring that encircles the DNA duplex
What is the function of the beta clamp?
Hold the polymerase onto the DNA while sliding along the duplex. Changes Pol III from distributive to processive enzyme, as it now stays attached to DNA during repetitive cycles of cNMP incorporation
Describe the structure of Pol III Core
Similar to Pol I
Palm (active site), Thumb (holds DNA), and finger (brings dNTPs)
What is the function of Pol III?
Pol III alpha subunit is the main replicative subunit and recruits E 3’-5’ proofreading activity
Descrine the structure of DNA Pol III Holoenzyme?
there are 3 Pol III core each with a beta sliding clamp. Each Pol III core attached to the clamp loader
Why does the holoenzyme have 3 DNA polymerases?
Facilitates the coordinated synthesis of the leading nad lagging strands at the replication fork
What assembles beta clamps onto the DNA strands?
Clamp loader
What is E.Coli beta clamp loader mechanism?
(1) ATP binds to t subunits indusing a conformational change that enables the clamp load to bind and open the clamp
(2) Binding ATP is necessary for clamp loader to bind DNA
(3) ATP hydrolysis causes the clamp loader to revert conformation and close - It cant bind beta clamp or DNA without ATP so it is ejected
Explain the trombone model of the replication fork
This refers to the lagging strand synthesis which is opposite of the replication fork. Pol III synthesizes from one Okazaki fragment to the next, which grows the loop (pulling out the trombone) when the next okazaki fragment is met it must detach from the beta clamp and associate with a new RNA primer close to the replication fork, which reduces the loop (pulling trombone back in)
Describe the recycling of Clamps
(1) Pol III dissociates from beta clamp
(2) Beta clamp site attracts Pol I, which is able to remove the RNA primer using its 5’3’ exonuclease activity
(3) Pol I dissociates from DNA, leaving ssDNA break. Beta clamp now attracts ligase to seal the break by forming phosphodiester bond using 3’OH and 5’P groups
(4) Beta clamp is then opened and unloaded by clamp loader
When can replication be terminated?
Once it’s started, there is no stopping it, it will complete.
What are the Ter sites of E.Coli
2 clusters of 23 base pair sequences located halfway around the circular chromosome, oriented in opposite directions
Describe the termination of E.Coli Chromosome Replication
Monomeric Tus protein tightly binds to a Ter site and blocks advance of the replication fork by stopping DnaB helicase
What does Tus stand for?
Termination utilization substance
What is an important property of the Tus-Ter complex
Its fork-blocking activity is directional = replication fork are blocked when approaching a Tus-Ter complex from 1 direction (non-permissive) but not when approaching from the opposite (permissive)
What effect does replication speed have on the Tus-Ter System
Tus-Ter system works regardless of speed, but replication forks of equal speed meet at the same time in terminus region, but not at unequal speed, the “fastest” one will be stuck in the terminus region until the slowest one is completed
Why is there competition between transcription and replication?
Actively replicating bacteria are also growing and metabolizing = actively transcribing RNA from promoters all over the chromosomes – this means that collisions between RNA polymerase and replication forks are inevitable
What happens when there is a codirectional collisions between RNA polymerase and transcription forks?
This does not impede the forks
What happens when there is a head on collisions between RNA polymerase and transcription forks?
Causes the fork to pause or stall
How is Competition minimized between transcription and replication?
Most transcripts in bacteria are oriented in the same direction as replication (codirectional) provided that the forks do not proceed more than halfway (would hit Tus-Ter System)
Describe what happens after both forks reach the terminus site
Results in the attachment of 2 daughter strands of DNA at the site of termination (Catenated daughter chromosomes) are unlinked into 2 separate chromosomes by action of type II topoisomerase enzyme
Describe the structure of topoisomerase in prokaryotes
Heterodimer with two ATPase domains and two cleavage core domains, linked by a scaffoliding domain
Describe the mechanism of action of topoisomerase in prokaryotes
(1) Topoisomerase pull one ot the pieces of catenated dsDNA into its cleavage site
(2) Uses ATP to generate a double-stranded break in this DNA fragment
Describe the mechanism of how topoisomerase can generate a double-stranded break
Tyr residue in catalytic site
(1) Tyr gives up its proton to form a nucleophile, which attacks the phosphodiester bond (occurs on both strands)
(2) This temporarily covalently links the DNA strand to topoisomerase by a phosphotyrosyl linkage (5’ adduct)
(3) Once daughter chromosomes are separated, hydrolysis reaction reforms the phosphodiester bonds
What is a 5’ adduct?
Forming of a phosphotyrosyl linkage between DNA and topoisomerase
Why does the End replication pose a problem in Eukaryotes?
The lagging strand has a single-stranded DNA gap that must be primed and filled in - due to when the RNA primer is removed for replacement, there is no 3’ terminus for DNA Pol to extend from, so cannot form dsDNA
The genetic information would be lost in the next round of replication
How is the end replication problem solved?
TERT-TR extends the 3’ terminus beyond what was replicated, so that a RNA primer can be added and the end can be transcripted
What are telomeres?
ends of linear eukaryotic chromosomes composed of repeats of a unique sequence
What is telomerase?
TERT-TR holoenzyme
When does the reaction of telomerase occur in the cell cycle?
S phase
Describe the mechanism of action of telomerase
(1) At the 3’-terminal end of a linear DNA, three nucleotides from the telomere links to three RNA nucleotides in telomerase
(2) Telomerase extends the 3’ end of the telomere by adding 1 telomere repeat (6 nucleotides)
(3) Telomerase repositions and repeats the last 2 steps many times, to add many telomere repeats
(4) The 3’ end that has been extended is converted to dsDNA by the same mechanism as chromosome replication (RNA primer, replisome, etc..) – because the end is a repeat that does not encode anything the cell can tolerate a certain amount of variability in final telomere length
(5) Still not completely duplex due to same RNA primer removal problem. Protected by telomere DNA-binding proteins so that they arent subjected to DNA repair mechanisms
