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BIOL253 > L4 + 5: Further DNA replication > Flashcards

L4 + 5: Further DNA replication Flashcards

1
Q

ori: bacteria vs euk, function of initiator proteins

A
  • 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
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2
Q

DnaA in bacteria

A
  • 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)
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3
Q

DnaB and DnaC interactions

A
  • 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
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4
Q

Eukaryotic replication origins - involvement of ORC

A
  • 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)
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5
Q

Influences on binding of ORC

A
  • Genetic features (G-rich seq, quadruplex etc)
  • Chromatin configuration (absence of nucleosomes)
  • Histone modification
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6
Q

Exception to euk. origin specificity

A

S.cerevisiae…
- Origin defined by specific sequence
- Yeast origins have 2 core seq (A, B1)
- A includes conserved 11 bp DNA seq - ARS

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7
Q

ORC binding stage 1 (pre-replicative complex)

A

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

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8
Q

ORC binding stage 2(GINS and the replicative complex)

A

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

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9
Q

Synthesising RNA primer: When is it carried out? Process in bacteria vs eukaryotes

A

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

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10
Q

Processivity in DNA pols

A
  • 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)
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11
Q

How do sliding clamps enhance processivity?

A
  • Slides along w/ the DNA pol, keeping it tethered
  • If the pol releases the 3’ OH of nascent strand, can’t dissociate so rebinds
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12
Q

Sliding clamp in euk

A

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

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13
Q

Sliding clamp in bacteria

A

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

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14
Q

Clamp loader basic structure, Detail for bacteria vs Eukaryotes

A

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

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15
Q

Clamp loader binding (discuss affinity with and without ATP)

A

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

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16
Q

Polymerase switching during clamp loading (euk)

A
  • 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
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17
Q

Coordination of leading and lagging strand

A
  • 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

18
Q

Okazaki Fragment maturation in E.coli

A
  • 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
19
Q

Okazaki fragment maturation in Euk

A

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

20
Q

Termination in bacteria

A
  • 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
21
Q

Topoisomerase involvement in bacterial DNA rep

A
  • Needed to unlink replicated circular chromosomes
  • Type IA can separate incompletely replicated molecules
    -Type II employed if replication has been completed (catenated loops)
22
Q

Relieving torsional stress

A
  • 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
23
Q

CMG complex and termination in Euk

A
  • 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
24
Q

The end-replication problem (2 contributing factors)

A

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

25
Q

Telomeres (end-replication problem)

A

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

26
Q

Telomerases; when do they act? Describe their structure

A
  • 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)
27
Q

How do telomerases stabilise chromosome ends?

A

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

28
Q

Telomere shortening in somatic cells

A
  • 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
29
Q

Overview of regulation of replication in E.coli

A
  • 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
30
Q

Regulatory inactivation of DnaA in E.coli (RIDA)

A

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)

31
Q

Regulation of inititaion of E.coli by methylation

A
  • 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
32
Q

Regulation of initiation in E.coli - DnaA availabilty

A
  1. 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
  2. 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
33
Q

Why/how is DNA replication more tightly controlled in euk vs bacteria

A
  • 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
34
Q

Origin timing vs efficiency

A
  • 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)
35
Q

Origin licensing by proteolysis

A
  • 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
36
Q

Preventing helicase loading outside G1.. Yeast

A
  • Export proteins from the nucleus
  • Also transcription inhibition and proteolysis for some proteins
37
Q

Preventing helicase loading outside G1… Metazoa i.e. humans

A
  • 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
38
Q

Chromatin replication maintenance

A
  • 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
39
Q

Recycling parental histones

A
  • 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
40
Q

Core histones in nucleosome replication

A
  • 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
41
Q

Synthesis and modification of new nucleosomes

A
  • 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

BIOL253 (19 decks)

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