L4 + 5: Further DNA replication Flashcards
ori: bacteria vs euk, function of initiator proteins
- Bacteria: 1 per chromosome
- Euk: initiate replication at many origins on each chromosome (much slower process, need to replicate simultaneously for it to occur in a timely fashion)
- Initiator proteins bind to origins and recruit helicases to unwind DNA (both bact, euk) -only when bound to ATP
- All initiator proteins are AAA+ ATPases
DnaA in bacteria
- DnaA binds to DnaA boxes in oriC
- When bound to ATP, self-associates into helical multi-subunit complex
- DNA wraps around spiral DnaA complex -> bending, local unwinding of AT rich sequence
- Then recruits helicase (DnaB)
- oriC (E.coli) has 245 bp, multiple DnaA boxes w/ adjacent AT rich region (a DNA unwinding element, fewer bonds)
DnaB and DnaC interactions
- DnaC helicase loader assembles onto the hexameric DnaB helicase
- DnaC binds to DnaA and places DnaB around ssDNA at origin - then dissociates
- Unwound DNA bound by SSB
Eukaryotic replication origins - involvement of ORC
- Much less known than in bacteria
- All bound by the initiator protein ORC (Origin Recognition Complex - 6 subunits, Orc1-6); doesn’t directly bind to DNA itself
- Generally not defined by specific sequences (can be initiated at different sites depending on Pr of being bound to ORC)
Influences on binding of ORC
- Genetic features (G-rich seq, quadruplex etc)
- Chromatin configuration (absence of nucleosomes)
- Histone modification
Exception to euk. origin specificity
S.cerevisiae…
- Origin defined by specific sequence
- Yeast origins have 2 core seq (A, B1)
- A includes conserved 11 bp DNA seq - ARS
ORC binding stage 1 (pre-replicative complex)
Origins are licensed in late M/G1 (prereplication complex established)…
- Cdc6, Cdt1 and ORC cooperate to load 2 ring-shaped MCM2-7 hexamers (sequentially, head-to-tail)
- ORC dissociates
- MCM complex inactive (in G1) - still encircles both strands
- MCM2-7 helicase = pre-replicative complex
ORC binding stage 2(GINS and the replicative complex)
Helicases activated in S phase…
- MCM2-7 complex phosph. by DDK, allows recruitment of Cdc45, Sld3
- S-phase CDK phosph., Sld2, Sld3 allowing GINS to be recruited
- Replicative helicase complex (CMG complex) - Cdc45-MCM-GINS
- Activated CMG helicase opens DNA (3’ to 5’ on leading strand)
-> goes from binding dsDNA to encircling ssDNA at origin
Synthesising RNA primer: When is it carried out? Process in bacteria vs eukaryotes
Carried out at origin (leading) or at start of each Okazaki fragment (lagging)
Bacteria
- Bacterial primase DnaG (single subunit RNA pol)
- Synth. RNA primer of 10-30 bases, then hands over to DNA pol III
Euk
- Polymerase alpha-primase complex (4 subunits)
- 2 subunit primase makes short RNA of 10 nt
- Polymerase alpha subunit adds short piece of DNA (iDNA/initiator DNA)
-> pol a has no proofreading, safest to limit to short piece of DNA
Processivity in DNA pols
- Can stay attached for many 1000s of nts
- Sliding clamp enhances processivity
e.g. DNA pol III 10bp/s vs 1000bp/s w/ clamp associated - In contrast, some DNA pols are distributive (add a few nts before falling off; poor/lack of proofreading)
How do sliding clamps enhance processivity?
- Slides along w/ the DNA pol, keeping it tethered
- If the pol releases the 3’ OH of nascent strand, can’t dissociate so rebinds
Sliding clamp in euk
PCNA (Proliferating Cell Nuclear Antigen)…
- interacts with DNA pol delta through motif of 8 AAs
- Ring consists if 3 PCNA pps (homotrimer), each w/ 2 similar domains
- Ring shape w/ 35 Angs. hole
Sliding clamp in bacteria
Beta protein…
- Part of DNA pol II holoenzyme
- Has a short peptide motif that interacts w/ bacterial DNA pol III core enzyme (which has alpha, epsilon subunits, with differing exonuclease activities, and are stabilised by theta subunit
- Ring consists of 2 beta subunits (homodimer), each w/ 3 similar domains
- Ring shape w/ 35 Angs. hole
Clamp loader basic structure, Detail for bacteria vs Eukaryotes
5 subunit clamp loader opens clamp, loads it onto DNA at prime template junction
Bacteria
- Gamma or Tau complex, 3 copies of y/T, one each of delta and delta’ - part of DNA pol III holoE
Euk
- Replication factor C (RFC)
- Has 5 different but related pps
Clamp loader binding (discuss affinity with and without ATP)
Loads sliding clamp onto 3’ end of RNA/iDNA primer…
- Low affinity for SC until bound to ATP
- Then binds to SC, forms spiral shape opening clamp (complex has high affinity for primer-template junction)
- Binding to junction stimulates ATPase activity
-> loader loses affinity for SC, released but SC remains
Polymerase switching during clamp loading (euk)
- Polymerase a-primase complex synth. RNA, initiator DNA -> dissociates
- Replaced by DNA pol delta or epsilon (handover = polymerase switching)
- Ensures replicative DNA pols are loaded onto DNA at right time and place to begin elongation
Coordination of leading and lagging strand
- Movement of leading, lagging strand DNA pols is coordinated at fork by looping round of lagging strand template
Bacteria
- Involves replisome (DnaB, DnaG primase, DNA pol III holoE)
- The DNA pols for each strand are tethered together via binding to flexible liner Tau subunits of clamp loader complex
- Ensures core enzyme remains associated at fork despite being released after each O frag.
Euk
- Ctf4 acts as a hub to couple CMG helicase, DNA pol epsilon and DNA pol a-primase at fork
- The 2 pols are not directly linked
Okazaki Fragment maturation in E.coli
- DNA pol III binds to RNA primer, extends, dissociates hen next RNA primer met (SC remains attached)
- DNA pol I recruited, removes RNA primer, fills gap w/ DNA
- Leaves nick in DNA which DNA ligase I seals
Okazaki fragment maturation in Euk
Promoted by Flap endonuclease (Fen1)…
- DNA pol delta continues to synth. DNA when it meets the RNA primer
- Displaces existing RNA primer and DNA from template strand, making a flap, which Fen1 cleaves
- DNA ligase then seals the nick
Termination in bacteria
- Occurs at ter sites within termination zone
- Each ter site is bound by a Tus molecule (stall the fork in one direction)
- terC most frequent (circular, 1st in clockwise direction)
- If anticlockwise fork encounters stalled fork, replication terminates as replisomes move past each other
- DNA pol I and DNA ligase I complete replication as termination completed
Topoisomerase involvement in bacterial DNA rep
- Needed to unlink replicated circular chromosomes
- Type IA can separate incompletely replicated molecules
-Type II employed if replication has been completed (catenated loops)
Relieving torsional stress
- Gets generated ahead of fork; must be removed (1 +ve supercoil for every DNA turn unwound)
- Topoisomerases (of all types) utilised for this purpose
- Supercoiling makes strand separation more difficult, particularly as two forks approach each other
CMG complex and termination in Euk
- Occurs at multiple sites in Euk
- When forks converge, CMG complexes move past each other on leading strand, CMG moves to encircle dsDNA
- DNA pol delta, Fen1 and DNA ligase 1 recruited to complete maturation
- dsDNA still entwined -> topoisomerase II employed
The end-replication problem (2 contributing factors)
1) RNA primer removal on the lagging strand leaves a gap that can’t be filled in
2) Dissolution of the replication fork when the lagging strand is finished, before lagging strand synthesis is complete - could lead to the loss of the terminal Okazaki fragment
-> Gradual erosion of Chr. ends w/ multiple rounds of replication
Telomeres (end-replication problem)
Ends protected by telomerases…
- Chr. ends have simple seq. repeats (TTAGGG in human)
-> Telomeres
- G-rich strand extends 5’ to 3’ towards Chr. end, terminates a ssDNA tail (~75-300nt)
-> forms unusual structure i.e. ‘T-loop’
- Telomeres mark the region as natural end of chromosome, distinguishing from a DNA break, serve as a landing pad for proteins that will protect the end maintaining chromosome stability
Telomerases; when do they act? Describe their structure
- Telomeres are maintained by telomerase (specialised reverse transcriptase)
- Active in tissue-spec. stem cells, germline cells. Synth. 1 strand of the telomere
- Has both protein and RNA components (Protein = Telomerase Reverse Transcriptase/TERT, RNA = template for synth of repeats)
How do telomerases stabilise chromosome ends?
Elongating the 3’ end of the telomere…
- RNA of telomerase base pairs w/ 3’ end of DNA
- Enzyme translocates and repeats synth. to elongate the g-rich 3’ end
- C-rich strand filled in by DNA pol-a
Telomere shortening in somatic cells
- Somatic tissues lack telomerase; telomere gradually shortens as cell divides
- This shortening activates the DNA damage response (Hayflick limit reached, run out of tandem repeats at the 3’ end))
- Telomerase expression re-activated in many cancers
Overview of regulation of replication in E.coli
- Regulated at level of initiation (coordinated w/ growth rate)
- Takes ~50 mins to replicate bacterial genome, so typically a second round is started before completion from origin
- Re-replication is coordinated at both oriC sites synchronously
-> no. oriC in a cell always a power of 2
Regulatory inactivation of DnaA in E.coli (RIDA)
Blocking replication initiation…
- DnaA binds and multimerises at oriC when bound to ATP, and unwinds adjacent DNA
- Helicase/primase/SC initiate synth.
- After initiation, SC stimulates hydrolysis of DnaA-ATP to ADP which then dissociates
-> can’t reinitiate origin firing (prevents overstimulation of replication initiation)
Regulation of inititaion of E.coli by methylation
- 11 GATC sites in oriC overlap the DnaA boxes, which are methylated by Dam methylase
- Before replication, GATC sequences are fully methylated - when this occurs, DnaA binds DnaA boxes at oriC
- After replication, DnaA boxes are transiently hemi-methylated
- SeqA binds to hemi-methylated GATC sites…
1. Prevents DnaA rebinding DnaA box
2. Prevents Dam methylase methylating at GATC
Regulation of initiation in E.coli - DnaA availabilty
- DNA sequestration
- DnaA can bind to datA DNA region adj. to oriC
- Sequesters 25% of free DnaA in cell
- After replication, datA region duplicated, twice as much sequestered, unavailable to rebind w/ oriC
- After segregation there’s only 1 copy of datA - Recycling of DnaA-ADP into DnaA-ATP
- DnaA binds to DARS region of DNA
- DARS serves as cofactors to stimulate exchange of ATP to ADP
Why/how is DNA replication more tightly controlled in euk vs bacteria
- Occurs only during S phase
- All DNA Must be duplicated exactly once
- Incomplete replication leads to inappropriate links btwn daughter cells
- Segregation causes breakage or loss
- Re-replication must be prevented
Origin timing vs efficiency
- Origin timing relates to when an origin fires ‘late’ or ‘early’
- Origin efficiency is the prob. an origin will fire
- Origin firing in S requires MCM loading to establish a pre-RC in G1; not every one will fire in every cell cycle, controlled by origin
- Most origin firing is stochastic (depends on replication protein availability)
Origin licensing by proteolysis
- In mammalian cells, origins are selected in G1 by..
1. ORC binding
2. Cdt1/Cdc6 mediated loading of MCM DNA helicase to form pre-RC - After loading, MCM DNA hel, Cdc6 and dt1 degraded
- In S phase, hel.s activate to initiate origin unwinding (once fire, can’t be reused until next G1)
- Cell cycle kinases involved in restricting initiation steps and origin firing to the appropriate timing
Preventing helicase loading outside G1.. Yeast
- Export proteins from the nucleus
- Also transcription inhibition and proteolysis for some proteins
Preventing helicase loading outside G1… Metazoa i.e. humans
- Proteolysis of proteins required to form pre-RC in S
- Geminin provides additional control; binds to Cdt1 and prevents it loading MCM during S, G and M phase
Chromatin replication maintenance
- Histone modifications must be maintained -epigenetic inheritance
- Synth of core histone subunits is tightly coupled to DNA synth - replication dependent histones or S phase histones
Recycling parental histones
- Nucleosomes are disrupted at replication fork by CMG helicase
- Parental histones are actively transferred to daughter DNA (parental histone segregation)
- Provides a guide to reconstruction of daughter chromatin w/ same modifications as parent
-> epigenetic inheritance
Core histones in nucleosome replication
- Parental H3 and H4 are distributed equally btwn daughters as tetramers (half to each daughter)
- Parental H2A and H2B are disassembled and reassembled on daughters as dimers
- Nucleosome disassembly and reassembly is mediated by histone chaperones, which interact w/ replication fork proteins to coordinate nucleosome disassembly and reassembly
Synthesis and modification of new nucleosomes
- Each daughter has half number of nucleosomes from parent
- New histones are synth. in S-phase (H3 and H4 histones acetylated on specific lysine residues in N-terminal tail)
- ASF1 and CAF1 promote assembly of new H3 and H4 histones into a tetramer and loading onto DNA (vi interaction w/ PCNA) to form half a nucleosome
- NAP1 and FACT add newly synth. H2A/H2B -> complete
- H3, H4 deacetylated
- Parental histones recruit histone modification enzymes to modify new histones
