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Chapt 21 DNA Replication II: Mechanisms; telomerase


Chapt 21 DNA Replication II: Mechanisms; telomerase Student learning outcomes: Describe how replication initiates proteins binding specific DNA sequences – PowerPoint PPT presentation

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Title: Chapt 21 DNA Replication II: Mechanisms; telomerase

Chapt 21 DNA Replication II Mechanisms
  • 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

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

E. Coli primosome Priming at oriC
  • 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

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

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

Point mutations define critical regions of SV40
ori AT regions T-Ag binding site
lt -Early genes
Late genes -gt
Fig. 21.4
ARS is Yeast Origin of Replication permit
replication of gene in yeast linker 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

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)

Processivity agent b-Subunit is clamp keeps pol
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.

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
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
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
b Clamp and g Clamp Loader
  • b-subunit needs help from g complex to load onto
  • 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

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
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
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
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

Decatenation Disentangling Daughter DNAs in
Fig. 27
  • End of replication, circular bacterial
    chromosomes are catenanes decatenated in 2
  • Melt unreplicated double-helical turns linking
    two strands
  • Repair synthesis fills in gaps
  • Decatenated by topoisomerase IV

Termination in Eukaryotes linear
chromosomes role 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
  • If no resolution, DNA strands get shorter each

  • Telomeres - special structures at ends of
  • One strand of telomeres is tandem repeats of
    short, G-rich regions (sequence varies among
  • G-rich telomere strand is made by enzyme
  • 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

  • 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
Telomere Formation telomerase makes DNA from RNA
template TERT, telomerase reverse
transcriptase proteins p43 and p123 1
RNA template Telomerase activity is high in
normal cells S phase, in cancer cells always
Telomere sequences vary Tetrahymena
TTGGGG Vertebrates TTAGGG Yeast
Fig. 31
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
  • 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

Mammalian Telomeres
  • T loop protects ss telomeric DNA (G-rich 3 end
  • 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
Review questions
  • 2. List the components of E. coli primosome and
    roles in primer synthesis.
  • 4. Outline strategy for identify yeast ARS
  • 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?