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Chapt 21 DNA Replication II: Mechanisms; telomerase

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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


1
Chapt 21 DNA Replication II Mechanisms
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

2
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 primosome Priming 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
7
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 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

10
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 agent b-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
15
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
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
19
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

20
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 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
    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
24
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
TTGGG
Fig. 31
25
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

26
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
27
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?
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