Lecture 4 - DNA Replication II Flashcards
Describe origins of replication in bacteria and eukaryotes.
Origins of replication are required to initiate replication in bacteria and eukaryotes.
* Origins are regions where the dsDNA is separated and replication initiated
* Bacteria have one origin per chromosome, eukaryotes initiate replication at many origins on each chromosome
* Initiator proteins bind to origins and recruit helicases to unwind the DNA
All initiator proteins are AAA+ ATPases
Describe initiation of DNA replication in bacteria.
1) In bacteria DnaA binding at oriC initiates local unwinding of DNA
* Bacterial origins are defined by specific DNA sequence to which the initiator protein DnaA binds.
* oriC – 245 bp sequence with multiple DnaA boxes (TTAT[C/A]CA[C/A]A) and adjacent (next to) to an AT rich region – a DNA unwinding element
DnaA proteins bind to DnaA boxes
When bound to ATP DnaA self-associates into a helical multi-subunit complex
DNA wraps around the spiral DnaA complex causing bending of DNA and local unwinding of adjacent AT rich sequence
2) The role of DnaB and DnaC
DNA wrapping around DnaA complex induces local unwinding of AT-rich region
DnaC helicase loader assembles onto the hexameric DnaB helicase (bind together)
DnaC helicase loader binds to DnaA bound at origin
DnaC helicase loader places DnaB helicase around ssDNA at origin. (completely encirculates the ssDNA)
DnaC loader dissociates from DnaB helicase
Unwound DNA is bound by SSB
Describe eukaryotic replication origins.
The unifying feature in eukaryotes is that the origins are bound by the initiator protein (origin Recognition Complex)
A few eukaryotes have origins defined by specific sequences (e.g. S. Cerevisiae) however most eukaryotic origins are not defined by specific sequences.
* Replication can be initiated at multiple different sites determined by the probability that they are bound by ORC
* Binding may be influenced by:
§ Genetic features e.g. G-rich sequence – quadruplex DNA
§ Chromatin configuration – absence of nucleosomes
§ Histone modification
Origin Recognition complex ORC – consists of 6 subunits (Orc1-6)
Describe origin unwinding in eukaryotes.
Origin unwinding occurs in 2 stages in eukaryotes
Origins are selected (Licensed) in late M/G1 – prereplication complex established
* ORC binds to DNA to establish origins
* Cdc6 and Cdt1 cooperate with ORC as DNA helicase loaders.
* Two ring-shaped MCM2-7 hexamers are loaded sequentially in a head-to-head orientation around dsDNA
* The ORC dissociates once the MCM2-7 pair is loaded
* In G1 the loaded MCM complex is inactive, remains encircling both DNA strands
* Loaded MCM2-7 helicase referred to as prereplicative complex
MCM- Mini Chromosome Maintenance
DNA helicases loaded around dsDNA at origins during G1 phase of cell cycle are activated in S-phase
* In S-phase the MCM2-7 complex is phosphorylated by DDK which allows recruitment of Cdc45 and Sld3
* S-phase CDK phosphorylates Sld2 and Sld3 allowing recruitment of GINS complex
* Replicative helicase complex – CMG complex (Cdc45-MCM-GINS)
* Activated CMG helicase opens DNA – transitions from binding ds DNA to encircling ssDNA at the origin – mechanism unknown
Describe priming of DNA synthesis during initiation of DNA replication.
- DNA replication can start once the origin has been opened and helicases loaded/activated
- DNA polymerases cannot initiate DNA synthesis de novo – they require an existing 3’OH for synthesis – provided by a primer.
- A primer is a short piece of RNA and is synthesized by DNA primase, a type of RNA polymerase
- Unlike DNA pols, RNA polymerases can synthesise de novo.
DNA primase synthesises RNA primers
* DNA replication can start once helicases are loaded.
* Primers must be synthesized on the leading strand only at origins
* On the lagging strand (discontinuous synthesis), primers must be synthesized to start each new Okazaki fragment
Bacterial Primase
* Bacterial primase DnaG is a single subunit RNA polymerase
* Synthesizes an RNA primer of 10-30 bases, then hands over to DNA polIII
Eukaryotic Polymerase
* Eukaryotic polymerase a-primase complex has 4 subunits
* A 2 subunit primase makes a short RNA of around 10 nucleotides.
The polymerase α subunit then adds a short piece of DNA - iDNA
Descrive the function of sliding clamps during Elongation of DNA replication.
DNA polymerases that replicate chromosomes are processive
* Processive synthesis
○ DNA polymerases that replicate entire chromosomes stay attached for many 1000s of nucleotides
○ Association with a sliding clamp processivity factor enhances processivity e.g. DNA pol III 10 bp/sec in absence of clamp, 1000 bp/sec in presence of clamp
* Non-processive or distributive synthesis
Some DNA pols with specialized functions add a few nucleotides before falling off - distributive
How do sliding clamps increase DNA polymerase processivity?
Sclamps increase DNA polymerase processivity
* The clamp binds DNA polymerase, slides along the DNA with the polymerase keeping it tethered to the DNA
* If the polymerase releases the 3’OH of the nascent strand it cannot dissociate away – rebinds the 3’OH and continues DNA synthesis
* PCNA interacts with eukaryotic DNA polymerase d through a motif of 8 amino acids in each PCNA subunit
* The b protein (part of the DNA pol III holoenzyme) has a short peptide motif that interacts with bacterial DNA pol III core enzyme
DNA pol III core enzyme – a, ε subunit
Describe the structure of both eukaryotic and bacterial sliding clamps.
Sliding Clamp (processivity factor) structure is conserved
* The high processivity of replicative DNA polymerase is due to association with a sliding clamp
* Bacterial sliding clamp is b protein - ring consists of two b subunits (homodimer) each with three similar domains
* Eukaryotes sliding clamp is Proliferating Cell Nuclear Antigen (PCNA) - ring consists of three PCNA polypeptides (homotrimer) each with two similar domains
Ring shape with 35 Å (3.5 nm) hole that encloses double stranded DNA
How are sliding clamps loaded?
Sliding clamps are loaded onto DNA by a clamp loader
* A 5-subunit clamp loader opens the clamp and loads it on to DNA at the prime template junction
○ g-complex in bacteria (aka t-complex), 3 copies of g (or t), one each d and d’ – part of the DNA polymerase III holoenzyme
○ Replication Factor C; RFC in eukaryotes – 5 different but related polypeptides
* Some of the clamp loader subunits are AAA+ ATPases – when ATP binds, the conformational change drives clamp loading
The clamp loader loads the sliding clamp on to the 3’ end of the RNA or iDNA primer
* The clamp loader has low affinity for the sliding clamp until the loader is bound to ATP
* On binding ATP, the clamp loader binds to the sliding clamp, forming a spiral shape and opening the clamp
* The clamp loader-sliding clamp complex has a high affinity with the primer-template junction
* Binding of the complex to the primer-template junction stimulates ATPase activity, which closes the sliding clamp and releases the clamp loader
* The sliding clamp remains associated with DNA
Sliding clamp recruits DNA pol to begin elongation
How does clamp loading facilitate polymerase switching?
- Polymerase a-primase complex synthesizes RNA and initiator DNA. Complex dissociates.
- Replaced by one of the processive eukaryotic DNA pols d or pol e
- Handover from Pol a-primase to DNA Pol d or DNA pol e is known as polymerase switching
- Replicative polymerase (d or e) is recruited by the sliding clamp
- DNA pol e replicates the leading strand and DNA pol d the lagging strand
Mechanism ensures that replicative DNA pols are loaded onto DNA at the right time and in the right place to begin elongation.
Describe the coordination of leading and lagging strand synthesis during elongation of DNA replication.
Leading and lagging strand synthesis are coupled at the replication fork
* DNA synthesis is towards direction of fork movement on leading strand and away from direction of fork movement on lagging strand.
* Movement of leading and lagging strand DNA pols is coordinated at the replication fork by looping round of the lagging strand template
Leading and lagging strand synthesis in bacteria is coordinated in the replisome
Replisome: DnaB helicase, DnaG primase, DNA polymerase III holoenzyme
Lagging and leading strand DNA polymerases are tethered together via binding to the flexible linker protein Tau subunits of the clamp loader complex.
Replisome ensures lagging strand DNA Pol III core enzyme remains associated at the fork even though it is released at the end of each Okazaki fragment.
Leading and lagging strand synthesis in eukaryotes is coordinated by Ctf4
Leading strand DNA polymerase e and lagging strand DNA polymerase d are both present at replication fork – but not directly linked - no equivalent of Tau protein
Multi-subunit Ctf4 acts as a hub to couple CMG helicase, DNA polymerase e and DNA polymerase a-primase at the fork
Describe the process of okazaki fragment maturation.
DNA is made in short discontinuous fragments on the lagging strand – Okazaki fragments
* Okazaki fragments are joined after synthesis
* DNA pol III disassociates from DNA when it reaches the next RNA primer.
* Sliding clamp remains attached
* DNA pol I is recruited and removes RNA primer (5’ to 3’ exonuclease activity), fills the gap with DNA
* DNA pol I leaves a “nick” in the DNA
* DNA ligase I seals the DNA “nick”
Flap endonuclease (Fen1) promotes Okazaki fragment maturation in eukaryotes
* DNA pol d continues to synthesise DNA when it meets the RNA primer
* This displaces the existing RNA primer and DNA from the template strand, making a flap
* Flap endonuclease (Fen1) cleaves the flap DNA
DNA ligase I seals the DNA “nick”
How is DNA replication terminated in bacteria.
- Termination of replication occurs at ter sites within the termination zone
- Each ter is bound by a single molecule of Tus
- ter sites bound by Tus stall the fork only in one direction
- terC used most frequently – first to be encountered (by clockwise fork)
- If anticlockwise fork encounters stalled clockwise fork at a Tus bound ter site, replication terminates as replisomes move past each other
- DNA polymerase I and DNA ligase I complete replication as termination completed.
Topoisomerases are required to unkink replicated circular chromosomes
* Type IA topoisomerases (which cleave one DNA strand and pass the other strand through the gap) can separate incompletely replicated molecules
Type II topoisomerases (which cleave both DNA strands of one DNA molecule) are required to separate completely replicated molecules
How is DNA replication terminated in eukaryotes?
Termination of DNA replication - eukaryotes
* DNA ahead of the fork gains one positive one positive supercoil for each DNA turn that is unwound
* Increased supercoiling ahead of the fork makes strand separation more difficult, especially as two replication forks approach one another
* Topoisomerases resolve the overwinding by transiently breaking DNA to remove supercoils allowing replication to be completed
* Termination of replication occurs at multiple sites in eukaryotes
* When replication forks converge, the CMG complexes move past each other on the leading strand, and CMG transitions to encircling dsDNA
* DNA polymerase d, Fen1 and DNA ligase 1 recruited to complete maturation (similar to Okazaki fragment processing)
Double-stranded DNA remains entwined, and this is resolved by a topoisomerase II .
