accessory factors summary 1.dna polymerase can’t replicate a genome. solutionatp? no single...
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Accessory factors summary
1. DNA polymerase can’t replicate a genome.Solution
ATP?No single stranded template Helicase
+The ss template is unstable SSB (RPA (euks))
-No primer Primase
(+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding
clamp - Sliding clamp can’t get on Clamp loader
(/RFC) +Lagging strand contains RNAPol I 5’-->3’ exo,
RNAseH -Lagging strand is nicked DNA ligase
+Helicase introduces + supercoils Topoisomerase II
+and products tangled
2. DNA replication is fast and processive
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DNA polymerase holoenzyme
QuickTime™ and aDV - PAL decompressor
are needed to see this picture.
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Maturation of Okazaki fragments
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Topoisomerases control chromosome topologyCatenanes/knots
Relaxed/disentangled
•Major therapeutic target - chemotherapeutics/antibacterials
•Type II topos transport one DNA through another
Topos
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Starting and stopping summary
1. DNA replication is controlled at the initiation step.
2. DNA replication starts at specific sites in E. coli and yeast.
3. In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader)
4. DnaA and DnaC reactions are coupled to ATP hydrolysis.
5. Bacterial chromosomes are circular, and termination occurs opposite OriC.
6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end.
7. Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome.
8. Telomerase extends the leading strand at the end.
9. Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits.
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Isolating DNA sequences that mediate initiation
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Different origin sequences in different organisms
E. Coli (bacteria)OriC
YeastARS(Autonomously Replicating Sequences)
Metazoans ????
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Initiation in prokaryotes and eukaryotesBacteria Eukaryotes
ORC + other proteins loadMCM hexameric helicases
MCM (helicase) + RPA (ssbp)
Primase + DNA pol
PCNA:pol
MCM (helicase) + RPA (ssbp)
PCNA:pol (clamp loader)
Primase + DNA pol
PCNA:pol DNA ligase
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Crystal structure of DnaA:ATP revealed mechanism of origin assembly
1. DnaA monomer (a) forms a polar filament (b).
2. DNA binding sites occur on the outside of the filament (model).
1. 2.
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Crystal structure of DnaA:ATP revealed mechanism of origin assembly
1. The arrangement of DNA binding sites introduces positive supercoils by wrapping DNA on the outside.
Compensating negative supercoils melt the replication bubble at the end.
2. Clamp deposition recruits Had, which promotes ATP hydrolysis and progressive disassembly of the DnaA filament (hypothesis).
1. 2.
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Initiation mechanism in bacteria -- 1
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Initiation mechanism in bacteria -- 2
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Initiation proteins in E. coli (bacteria)
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10 ter sites opposite oriC coordinate the end game
The ter/tus system is not essential in E. coli.
Tus protein binds Ter sites and inhibits the DnaB helicase
OriginCounterclockwise
forkClockwisefork
Clockwisefork trap
Counterclockwisefork trap
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Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”
Releasing C6 springs the trap
DNA Half life (s) Kd (nM)
130 (2 min) 1.6
53 (<1 min, FAST/ 53
permissive)
6900 (115 min, SLOW/ 0.4
nonpermissive)
terB
C6
C6
C6
Mulcair et al. (2006) Cell 125, 1309-1319.
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Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”
Releasing C6 springs the trap
DNA Half life (s) Kd (nM)
130 (2 min) 1.6
53 (<1 min, FAST/ 53
permissive)
6900 (115 min, SLOW/ 0.4
nonpermissive)
terB
C6
C6
C6
5’3’
Mulcair et al. (2006) Cell 125, 1309-1319.
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Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”
Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.
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Unwinding ter from the nonpermissive direction springs a “molecular mousetrap”
Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.
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Topoisomerase II unlinks the replicated chromosomes
Topoisomerase II - Cuts DNA and passes one duplex through the other.
Class II topoisomerases include:Topo IV and DNA gyrase
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Summary: What problems do these proteins solve?
Tyr OH attacks PO4 and forms a covalent intermediate
Structural changes in the protein open the gap by 20 Å!
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Function E. coli SV40 (simian virus 40)
Helicase DnaB T antigen
Primase
Primer removal
Primase (DnaG)
pol I’s 5’-3’exo
pol primase
FEN 1 (also RNaseH)
Polymerase
Core pol III (, , subunits) pol ,
Clamp loader complex RF-C
Sliding clamp PCNA
ssDNA binding SSB RF-A
Remove +sc at fork (swivel) gyrase topo I or topo II
Decatenation topo IV topo II
Ligase DNA ligase DNA ligase I
… other model systems include bacteriophage T4 and yeast
Summary: What problems do these proteins solve?
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The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome.
Not enough nucleotides for primase to start last lagging strand fragment
Chromosome ends shorten every generation!
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Telomere shortening signals trouble!
1. Telomere shortening releases telomere binding proteins (TBPs)
2. Further shortening affects expression of telomere-shortening sensitive genes
3. Further shortening leads to DNA damage and mutations.
Telomere binding proteins (TBPs)
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Telomerase replicates the ends (telomeres)
Telomere ssDNA
Telomerase extends the leading strand!Synthesis is in the 5’-->3’ direction.
Telomerase is a ribonucleoprotein (RNP). The enzyme contains RNA and proteins.
The RNA templates DNA synthesis. The proteins include the telomerase reverse transcriptase TERT.
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Telomerase cycles at the telomeres
Telomere ssDNA
TERT protein
TER RNA template
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Telomerase extends a chromosome 3’ overhang
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Conserved structures in TER and TERT
Core secondary structures shared in ciliate and vertebrate telomerase RNAs (TERs). (Sequences highly variable.)
148-209 nucleotides
1000s of nucleotides
TERT protein sequences conserved
1300 nucleotides
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Starting and stopping summary
1. DNA replication is controlled at the initiation step.
2. DNA replication starts at specific sites in E. coli and yeast.
3. In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader)
4. DnaA and DnaC reactions are coupled to ATP hydrolysis.
5. Bacterial chromosomes are circular, and termination occurs opposite OriC.
6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end.
7. Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome.
8. Telomerase extends the leading strand at the end.
9. Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits.