Title: Chapt 21 DNA Replication II: Mechanisms; telomerase
1Chapt 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
221.1 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
3E. 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
4Key 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
5Priming 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
6Replication 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
7Origin 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
8Point mutations define critical regions of SV40
ori AT regions T-Ag binding site
lt -Early genes
Late genes -gt
Fig. 21.4
9ARS 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
1021.2 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)
11Processivity 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)
12Pol 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
13DNA 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
14Eukaryotic 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
15b 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
16Pol 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
17Simultaneous 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
18Lagging 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
1921.3 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
20Decatenation 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
21Termination 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
22Telomeres
- 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
24Telomere 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
25Telomere 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
26Mammalian 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
27Review 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?