DNA Replicates by a Semiconservative Mechanism

1
DNA Replicates by a Semiconservative Mechanism
Grow cells in 15N and transfer to 14N
Analyze DNA by equilibrium density gradient
centrifugation
Presence of H-L DNA is indicative of
semiconservative DNA replication
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-329
2
The Golden Rule of DNA Replication
3
Mechanics of DNA Replication in E. coli
Leading strand is synthesized continuously and
lagging strand is synthesized as Okazaki
fragments
The 5 to 3 exonuclease activity of Pol I
removes the RNA primer and fills in the gap
DNA ligase joins adjacent completed fragments
from Lodish et al., Molecular Cell Biology, 4th
ed. Fig 12-9
4
Initiation of DNA Replication in E. coli
DnaA binds to 9-mers at oriC and melts the
13-mer segments
DnaC brings DnaB hexamer to single stranded
regions
DnaB helicase unwinds the DNA away from the
origin
from Lodish et al., Molecular Cell Biology, 4th
ed. Fig 12-7
5
DnaB is an ATP-dependent Helicase
DnaB unwinds DNA in the 5 to 3 direction
DnaB uses ATP hydrolysis to separate the strands
SSB proteins prevent the separated strands from
reannealing
from Lodish et al., Molecular Cell Biology, 4th
ed. Fig 12-8
6
RNA Primer Synthesis Does Not Require a 3-OH
Primase is recruited to ssDNA by a DnaB hexamer
from Alberts et al., Molecular Biology of the
Cell, 4th ed., Fig 5-12
7
Coordination of Leading and Lagging Strand
Synthesis
Two molecules of Pol III are bound at each
growing fork and are held together by t
The size of the DNA loop increases as lagging
strand is synthesized
Lagging strand polymerase is displaced when
Okazaki fragment is completed and rebinds to
synthesize the next Okazaki fragment
from Lodish et al., Molecular Cell Biology, 4th
ed. Fig 12-11
8
Bidirectional Replication of SV40 DNA from a
Single Origin
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-32
9
Replication of SV40 DNA
T antigen binds to origin and melts duplex and
RPA binds to ss DNA
Primase synthesizes RNA primer and Pol a extends
the primer
PCNA-Rfc-Pol d extend the primer
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 4-31
10
Replication Origins in Eukaryotes
Origins contain specific sequences favorable for
initiating DNA replication that bind ORC
Replication will initiate within any DNA
sequence in many eukaryotic systems
Origin choice becomes more specific during
development
from Gilbert, Science 294, 96 (2001)
11
Assembly of preRC During Licensing
ORC is recruited to oris
ORC recruits Cdc6 and Cdt1, and the complex
loads Mcm2-7 hexamers
Association of two Mcm2-7 complexes form
initiation-competent complex
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6,
476 (2005)
12
Replication Origins are Licensed in Late M and G1
Origins are licensed by Mcm2-7 binding to form
part of the pre-RC
Mcm2-7 is displaced as DNA replication is
initiated
Licensing is turned off at late G1 by CDKs
and/or geminin
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6,
476 (2005)
13
Control of Licensing Differs in Yeasts and
Metazoans
CDK activity prevents licensing in yeast
Geminin activation downregulates Cdt1 in
metazoans
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6,
476 (2005)
14
Roles and Actions of Telomeres
End replication problem - Removal of RNA primer
at the end of lagging strand leaves a gap and
can lead to progressive loss of terminal sequences
Cells must distinguish natural chromosome ends
from sites of DNA damage to prevent checkpoint
activation
Telomerase creates an array of short repeats at
chromosome ends
Telomere-specific proteins bind to repeats
15
Solutions to the End-replication Problem
3-terminus is extended using the reverse
transcriptase activity of telomerase
Dipteran insects use retrotransposition with the
3-end of the chromosome as a primer
Kluyveromyces lactis uses a rolling circle
mechanism in which the 3-end is extended on an
extrachromosomal template
Telomerase-deficient yeast use a
recombination- dependent replication pathway in
which one telomere uses another telomere as a
template
Formation of T-loops using terminal repeats
allow extension of invaded 3-ends
from de Lange, Nature Rev.Mol.Cell Biol. 5, 323
(2004)
16
Mechanism of Telomerase Action
Telomerase RNA anneals to ss 3terminus
dNTPs are added by the reverse transcriptase
activity of telomerase using telomerase RNA as a
template
Repeated displacement of the DNA-RNA duplex and
reverse transcription lengthens the telomere
New Okazaki fragments are synthesized on the
extended template strand
from Alberts et al., Molecular Biology of the
Cell, 4th ed. Fig 5-43
17
Shelterin Specifically Associates with Telomeres
Shelterin subunits specifically recognize
telomeric repeats
Shelterin allows cells to distinguish telomeres
from sites of DNA damage
Shelterin may inhibit the activation of the DNA
damage pathway by inhibiting ATM
from de Lange, Genes Dev. 19, 2100 (2005)
18
Telomere Termini Contain a 3-Overhang
A nuclease processes the 5-end
POT1 controls the specificity of the 5-end
from de Lange, Genes Dev. 19, 2100 (2005)
19
Formation of the t-Loop
TRF1 binds ds telomeric repeats
TRF1 contains DNA bending and looping activity
TIN2 enhances the architectural effects of TRF1
TRF2 recruits the MRE11 complex to promote
strand invasion
from de Lange, Genes Dev. 19, 2100 (2005)
20
Telomerase Action is Restricted to a Subset of
Ends
Telomere length is regulated by shelterin
Increased levels of shelterin inhibits telomerase
action
Telomerase is inhibited by increased amounts of
POT1
Elongation of shortened telomeres depends on the
recruitment of the Est1 subunit of telomerase by
Cdc13 end-binding protein
from Bertuch and Lundblad, Curr.Opin.Cell Biol.
18, 247 (2006)
21
Dysfunctional Telomeres Induce the DNA Damage
Response
Shelterin may contain an ATM inhibitor
Telomere damage activates ATM
DNA damage response protein accumulate at
unprotected telomeres
ATM activates p53 and leads to cell cycle arrest
or apoptosis
from de Lange, Genes Dev. 19, 2100 (2005)
22
T-loops are Similar to the Initiation of the RDR
Pathway
Some telomere-associated proteins play a role in
RDR
from de Lange, Nature Rev.Mol.Cell Biol. 5, 323
(2004)
23
Model for Evolution of Telomeres
The first linear chromosomes may have acquired
terminal repeats
Terminal repeats may have been capped and
maintained by t-loops
Telomerase may have evolved from a pre-existing
retrotransposon RTase
T-loops are not used for telomere replication
from de Lange, Nature Rev.Mol.Cell Biol. 5, 323
(2004)