Chapt 21 DNA Replication II: Mechanisms; telomerase

1
Chapt 21 DNA Replication IIMechanisms
telomerase

• Student learning outcomes
• Describe how replication initiates
• proteins binding specific DNA sequences
• Explain general elongation
• coordination leading, lagging strands
• Describe basic features of termination
• Describe basic features of telomerase
• Important Figs 1, 7, 11, 18, 19, 25, 26, 29,
  30, 31, 32, 34, 36
• Review problems 1-5, 8, 11, 14, 16, 21, 22, 23,
  24, 25

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21.1 Initiation and Priming in E. coli

• Initiation of DNA replication requires primers
• Different organisms use different mechanisms
  for primers
• Primosome - - proteins needed to make primers to
  replicate DNA (A. Kornberg)
• E. coli primosome
• DNA helicase (DnaB)
• Primase (DnaG)
• Primosome assembly at origin of replication, oriC
  is multi-step

3
E. Coli primosomePriming at oriC
primase

• DnaA binds to unique oriC at sites called dnaA
  boxes cooperates with RNAP, HU protein to melt
  nearby DNA region
• DnaB binds to open complex, facilitates
  binding of primase to complete primosome
• DnaB helicase activity unwinds DNA
• Primosome remains with replisome, repeatedly
  primes Okazaki fragment synthesis on lagging
  strand

4
Key proteins at the DNA replication fork
Figure 6.13 of Hartl Jones Role of key
proteins in DNA replication draw 5 and 3
ends, leading, lagging strands
5
Priming in Eukaryotes

• Eukaryotic replication is more complex
• Bigger size of eukaryotic genomes, most are
  linear
• Slower movement of replicating forks
• Each chromosome must have multiple origins
• Model monkey virus SV40 (5200 nt genome)
• Later yeast ARS (centromere) regions

6
Replication of SV40 is bidirectional Isolate
replicating molecules cleave with EcoRI that has
1 site look at molecules in EM (A-j increasing
replication).
Fig. 21.2
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Origin of Replication in SV40

• SV40 ori adjacent to transcription control region
• Initiation of replication needs viral large T
  antigen (major product of early transciption)
  binding to
• Region within 64-bp ori core
• Two adjacent sites
• T antigen helicase activity opens up replication
  bubble within ori core
• Priming carried out by primase associated with
  host DNA pol a

8
Point mutations define critical regions of SV40
ori AT regions T-Ag binding site
lt -Early genes
Late genes -gt
Fig. 21.4
9
ARS is Yeast Origin of Replication permit
replication of gene in yeastlinker scanning
mutants define critical regions plasmid has
centromere, URA gene grow non-selective and then
check Ura (Fig. 7)

• Autonomously replicating sequences (ARSs)
• 4 important regions
• Region A - 15 bp long with11-bp consensus
  sequence highly conserved in ARSs
• B1 and B2
• B3 may permits important DNA bend within ARS1

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21.2 Elongation and processivity

• Once primer in place,DNA synthesis begins
• Coordinated synthesis of lagging and leading
  strands keeps pol III holoenzyme on template
• Replication is highly processive, very rapid
• pol III holoenzyme in vitro 730 nt/sec (in
  vivo 1000 nt/sec)
• Pol III core alone is poor polymerase after 10
  nt it falls off
• Takes time to reassociate with template and
  nascent DNA
• Missing from core enzyme is processivity factor
• sliding clamp, b-subunit of holoenzyme (see
  Table 20.2)

11
Processivity agentb-Subunit is clamp keeps pol
III on DNA
Fig. 12 b-dimer on DNA

• Core plus b-subunit replicates DNA processively
• ( 1,000 nt/sec)
• Dimer formed by b-subunit is ring-shaped
• Ring fits around DNA template
• Interacts with a-subunit of core to tether whole
  polymerase and template (Fig. 9)
• Holoenzyme stays on template with b-clamp (Fig.
  11)

12
Pol III Holoenzyme Table 20.2
Pol III core has 3 subunits Pol III g complex
has 5 subunits DNA-dependent ATPase Pol III
holoenzyme includes b subunit
13
DNA pol III subunits bind each other a, e core
g ATPase, b clamp Purified subunits mixed and
chromatographed to separate complexes from free
proteins SDS-PAGE Western blot tests which
proteins in which complexes Also assayed DNA
polymerase activity
Fig. 21.10
14
Eukaryotic processivity factor

• PCNA forms trimer, a ring that encircles DNA and
  holds DNA polymerase on the template

Fig. 14
Fig. 13 b-dimer on DNA in E. coli
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b Clamp and g Clamp Loader

• b-subunit needs help from g complex to load onto
  DNA
• This g complex acts catalytically to form
  processive adb complex
• g not remain associated with complex during
  processive replication
• Clamp loading is ATP-dependent
• Energy from ATP changes conformation of loader so
  d-subunit binds one b-subunits
• Binding opens clamp, allows it to encircle DNA

16
Pol III subassembly has 2 cores, one g and no b

• Recall from table 2
• Core pol III has 3 subunits
• is polymerase
• is exonuclease
• t dimerizes core

Fig. 17 g complex has 5 subunits
17
Simultaneous Strand Synthesis by double-headed
pol III

• 2 core polymerases attached through 2 t-subunits
  to g complex
• One core responsible for continuous synthesis of
  leading strand
• Other core performs discontinuous synthesis of
  the lagging strand
• g complex serves as clamp loader to load b clamp
  onto primed DNA template
• After loading, b clamp loses affinity for g
  complex instead associates with core polymerase

Fig. 18
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Lagging Strand Replication

• g complex and b clamp help core polymerase with
  processive synthesis of Okazaki fragment
• When fragment completed, b clamp loses affinity
  for core
• b clamp g complex acts to unload clamp
• Now clamp recycles

Fig. 25
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21.3 Termination of replication
Fig. 26

• Straightforward for phage like l that produce
  long, linear concatemers (rolling circle)
• Grows until genome-sized piece cut off, packaged
  into phage head
• Bacterial replication 2 replication forks
  approach each other at terminus region
• 22-bp terminator sites bind specific proteins
  (terminus utilization substance, TUS)
• Replicating forks enter terminus region, pause
• 2 daughter duplexes entangled, must separate

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Decatenation Disentangling Daughter DNAs in
Bacteria
Fig. 27

• End of replication, circular bacterial
  chromosomes are catenanes decatenated in 2
  steps
• Melt unreplicated double-helical turns linking
  two strands
• Repair synthesis fills in gaps
• Decatenated by topoisomerase IV

21
Termination in Eukaryoteslinear
chromosomesrole of telomerase
Fig. 29

• Eukaryotes have problem filling gaps left when
  RNA primers are removed after DNA replication
• DNA cannot be extended 3?5 direction
• No 3-end upstream (unlike circular bacterial
  chromosome)
• If no resolution, DNA strands get shorter each
  replication

22
Telomeres

• Telomeres - special structures at ends of
  chromosomes
• One strand of telomeres is tandem repeats of
  short, G-rich regions (sequence varies among
  species)
• G-rich telomere strand is made by enzyme
  telomerase
• Telomerase contains a short RNA that is template
  for telomere synthesis
• C-rich telomere strand is synthesized by ordinary
  RNA-primed DNA synthesis
• Process like lagging strand DNA replication
• Ensures chromosome ends are rebuilt, do not
  suffer shortening each round of replication

23

• Tetrahymena cells have telomerase activity
• Greider Blackburn
• Cell extracts, synthetic oligo 32P-dNTPs,
  other dNTP
• Conclusion
• enzyme adds 6-bp units
• Only needs GTP, TTP
• (lanes 3, 6)
• template (TTGGGG)4

Fig. 21.30
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Telomere Formationtelomerase makes DNA from RNA
templateTERT, telomerase reverse
transcriptase proteins p43 and p123 1
RNA templateTelomerase activity is highin
normal cells S phase, in cancer cells always
Telomere sequences vary Tetrahymena
TTGGGG Vertebrates TTAGGG Yeast
TTGGG
Fig. 31
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Telomere Structure
Fig. 32

• Eukaryotes protect telomeres from nucleases and
  ds break repair enzymes
• Ciliates have TEBP (telomere end-binding protein)
  to bind and protect 3-single-strand telomeric
  overhang
• Budding yeast has Cdc13p which recruits Stn1p and
  Ten1p that all bind ss telomeric DNA
• Mammals and fission yeast have protein similar to
  TEBP binding to ss telomeric DNA

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Mammalian Telomeres

• T loop protects ss telomeric DNA (G-rich 3 end
  loops)
• Proteins TRF1 and TRF2 help telomeric DNA form
  loop in which ss 3-end of telomere invades ds
  telomeric DNA
• TRF1 may bend DNA into shape for strand invasion
• TRF2 binds at point of strand invasion, may
  stabilize displacement loop

Fig. 36
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Review questions

• 2. List the components of E. coli primosome and
  roles in primer synthesis.
• 4. Outline strategy for identify yeast ARS
  sequence.
• 14. How can discontinuous synthesis of lagging
  strand keep up with continuous synthesis of
  leading strand?
• 21. Why do eukaryotes need telomeres, but
  prokaryotes do not?