The initiation of yeast DNA replication

1
BMB801 Lecture 16 -- Dr. Michael Weinreich
10/4/06
The initiation of yeast DNA replication Quest
ions? Contact michael.weinreich_at_vai.org Van Andel
Research Institute Grand Rapids, MI 49503
2
Initiation of DNA Replication
Essential for cell growth, development, integrity
of the genetic information Each origin initiates
replication only once per cell cycle Highly
regulated. Commitment to DNA replication is an
irreversible decision Many initiation proteins
are upregulated in cancer cells
3
Initiation of DNA Replication
Essential for cell growth, development, integrity
of the genetic information Each origin initiates
replication only once per cell cycle Highly
regulated. Commitment to DNA replication is an
irreversible decision Many initiation proteins
are upregulated in cancer cells

• QUESTIONS
• What are the requirements for the initiation of
  DNA replication?
• How is initiation limited to once per cell cycle?
• What effect might chromatin structure have on
  initiation?
• Overview of the temporal nature of replication
  initiation

4
DNA Replication Begins at Specific Sequences
Fiber Autoradiography Visualize 3H-Thymidine
incorporation following a short pulse
5
Isolation of Autonomously Replicating Sequences
origin
Restriction digest of yeast chromosomal DNA
Shotgun clone into a URA3 origin-less plasmid
Transform into ura3 yeast
Select for URA cells
6
Isolation of Autonomously Replicating Sequences
EcoRI
URA3
URA3
URA3
EcoRI
URA
7
Isolation of Autonomously Replicating Sequences
EcoRI
URA3
URA3
URA3
EcoRI
8
Isolation of Autonomously Replicating Sequences
EcoRI
URA3
URA3
URA3
EcoRI
Ura-
9
Isolation of Autonomously Replicating Sequences
EcoRI
URA3
URA3
URA3
EcoRI
URA
-URA plate
10
ARS elements consist of two domains
ARS element
150-200bp
11
ARS elements share a common sequence - the ACS
ARS element
150-200bp
WTTTAYRTTTW
ARS Core Consensus Sequence
W A or T R A or G Y T or C
12
Plasmid stability measurements of linker scan
mutants
ARS element
GGTCGAC
150-200bp
SalI
1. Transform plasmid into yeast selecting for
URA3 marker Failure to recover high frequency
of transformation (HFT) - essential sequences 2.
Grow yeast in medium lacking uracil to get a
population of cells 3. Grow in medium containing
uracil to allow plasmid loss events 4. Calculate
percentage of cells containing plasmid after X
generations
13
Linker scan analysis of ARS315
B1
A
B2
IS
Percentage of plasmid containing cells
_ _
HFT



ACS
14
Linker scan analysis reveals modular structure of
origins
A
B1
B2
B3
ARS1
ARS307
ACS
Important
Essential
15
There are at least two broad classes of origins
in S. cerevisiae
A
B1
B2
B3
ARS1
A
B1
B2
IS
ARS315
ARS305
ARS307
?
ACS
Inhibitory element
16
Jacob and Brenners Replicon Model
Initiator protein
Replicator
WTTTAYRTTTW
Jacob, F., and S. Brenner. 1963. On the
regulation of DNA synthesis in bacteria the
hypothesis of the replicon. C R Hebd Seances
Acad Sci 256 298-300.
17
Jacob and Brenners Replicon Model
Initiator protein -- DnaA
Replicator -- OriC
13mers
DnaA boxes
E. coli
18
Jacob and Brenners Replicon Model
Initiator protein -- ORC (Origin Recognition
Complex)
A
B1
B2
B3
Replicator -- ARS
Bell, SP and Stillman, B. (1992) ATP-dependent
recognition of eukaryotic origins of DNA
replication by a multiprotein complex. Nature
357(6374)128-34.
19
ORC binds to origins of replication
ORC
A B1 B2
origin
-ORC is a six-subunit protein complex that binds
to ARS elements. -Specific DNA binding requires
ATP -All subunits are essential and are required
for initiation -ORC binds ARS elements
throughout the cell cycle
20
and then recruits Cdc6p and Cdt1p during early G1
Cdc6p
Cdt1p
ORC
ORC
A B1 B2
A B1 B2
origin
21
Cdc6 is a AAA protein required for pre-RC
assembly
K114A mutant is ts cdc6-4
22
The pre-Replicative Complex assembles at all
origins in G1
MCM
ORC
A B1 B2
pre-RC
23
Kinases then activate the initiation of DNA
synthesis
MCM
ORC
ORC
A B1 B2
A B1 B2
Cdc45p
GINS
Cdc7p-Dbf4p
pre-RC

Cdk1-Clb
Ser/Thr protein kinases
24
Origin unwinding occurs
MCM
ORC
A B1 B2
Cdc45p
GINS
25
followed by the assembly of DNA polymerases
and bi-directional DNA synthesis
26
P
Cdt1
P
Cdk Kinase
27
How do cyclin-dependent kinases prevent pre-RC
formation?
CyclinB-Cdks phosphorylate ORC, Cdc6p and MCM
proteins ORC phosphorylation inhibits its
activity through an unknown mechanism Cdc6p
phosphorylation causes is proteolysis MCM
phosphorylation excludes it from the nucleus, as
well as Cdt1p
P
P
P
MCM
ORC
ORC
A B1 B2
A B1 B2
High Cdk levels
pre-RC Low Cdk levels
28
P
Cdt1
P
Cdk Kinase
29
ORC and Abf1p position nucleosomes outside ARS1
Abf1p
ORC
A
B1
B2
B3
30
facilitating pre-RC formation at the origin
Cdc6p
Cdt1p
Abf1p
MCM
ORC
A
B1
B2
B3
31
Origins lacking a B3 element may be sensitive to
nucleosome intrusion
Cdc6p
Cdt1p
Abf1p
MCM
ORC
A
B1
B2
B3
ORC
A
B1
B2
32
that inhibits pre-RC assembly
Cdc6p
Cdt1p
Abf1p
MCM
ORC
A
B1
B2
B3
Cdc6p
Cdt1p
ORC
A
B1
B2
33
IS element may position a nucleosome over origin
A
B1
B2
B3
ARS1
A
B1
B2
IS
ARS315
nucleosome
ARS305
ARS307
?
ACS
34
Position of ARS elements along chromosome III
302 303 320
314
318
301
308
300
304
305
306
309
310
313
315
316
317
319
307
Chromosome III
lt 10
Inactive
lt 10
35
Position of ARS elements along chromosome III
Early origins
302 303 320
314
318
301
308
300
304
305
306
309
310
313
315
316
317
319
307
Chromosome III
lt 10
Inactive
lt 10
36
Position of ARS elements along chromosome III
Early origins
302 303 320
314
318
301
308
300
304
305
306
309
310
313
315
316
317
319
307
Chromosome III
lt 10
Inactive
lt 10
Late replicating
Telomeres
37
Replication timing along chromosome VI
Raghuraman MK et al. (2001) Replication dynamics
of the yeast genome Science 294(5540)115-21.
38
(No Transcript)
39
BMB801 Lecture 17 -- Dr. Michael Weinreich
10/6/06
DNA damage during S-phase, telomeres, late
replication and the centromere Questions? Cont
act michael.weinreich_at_vai.org Van Andel Research
Institute Grand Rapids, MI 49503
40
Linker scan analysis reveals modular structure of
origins
A
B1
B2
B3
ARS1
ARS307
ACS
Important
Essential
41
Plasmid stability measurements of linker scan
mutants
ARS element
150-200bp
Linker scans are typically 6-10bp in length.
They change the existing sequence at multiple
base pairs without the adding or deleting
nucleotides and introduce a novel restriction
site. For example, an 8bp XhoI linker scan across
this region might change the existing sequence to
CCTCGAGG at each position above.
42
Formation of the pre-RC occurs during G1
Cdc6p
MCM
Cdt1p
ORC
ORC
ORC
A B1 B2
A B1 B2
A B1 B2
origin
MCM, DNA helicase
ORC, Origin Recognition Complex
Cdc6p and Cdt1p, helicase loaders
43
Kinases then activate the initiation of DNA
synthesis
MCM
ORC
ORC
A B1 B2
A B1 B2
Cdc45p
GINS
Cdc7p-Dbf4p
pre-RC

Cdk1-Clb
Ser/Thr protein kinases
44
by promoting the assembly of DNA polymerases
and bi-directional DNA synthesis
45
Late replication, the telomere and functions of
the centromere
QUESTIONS 5. What determines the temporal order
of replication during S-phase? 6. How might DNA
damage affect DNA replication? 7. What are
telomeres and how are they replicated? 8.
Overview of the centromere and kinetochore
46
The pre-Replicative Complex assembles at all
origins in G1
MCM
ORC
Early and late origins assemble the pre-RC during
G1 Since some origins are not activated until
late S-phase, the regulatory step must occur
after pre-RC formation Cdc7-Dbf4 and
Cdk1-Clb5,6?
A B1 B2
pre-RC
47
Replication timing along chromosome VI
Raghuraman MK et al. (2001) Replication dynamics
of the yeast genome Science 294(5540)115-21.
48
MMS activates the intra-S-phase checkpoint which
inhibits late origin firing
DNA damage (MMS)
Rad9p
Mec1p (ATR)
Mec2/Ddc1 (ATRIP)
Rad53p (Chk2)
Chk1p
Inhibit late origin firing
G2/M
origin
WT
49
MMS activates the intra-S-phase checkpoint which
inhibits late origin firing
DNA damage (MMS)
Rad9p
Mec1p (ATR)
Mec2/Ddc1 (ATRIP)
Rad53p (Chk2)
Chk1p
Inhibit late origin firing
G2/M
origin
WT
MMS
Slows S-phase
50
Cdc7p-Dbf4p
Dbf4p is phosphorylated following replication
arrest
P
Dbf4p-Cdc7p
?
pre-RC
Late origins
Rad53p
Mec1p
DNA damage Replication arrest
51
Cdc7p-Dbf4p
Homologous proteins in S. pombe, Xenopus, mouse
and humans Cdc7p is a serine/threonine kinase
required for entry into S phase after assembly
of the pre-RC Dbf4p is a regulatory subunit
required for kinase activity Phosphorylates MCM
proteins, Cdc45p and polymerase-a primase in
vitro Cdc7p-Dbf4p is required for loading Cdc45p
and GINS at the origin
MCM
ORC
ORC
A B1 B2
A B1 B2
Cdc45p
GINS
Cdc7p-Dbf4p
pre-RC

Cdk1-Clb
52
DBF4 contains three regions of homology among
diverse species
Dbf4p
C
N
M
704
1
350
Is the N-terminus required for viability (DNA
replication) or DNA repair?
What role might Dbf4p play in checkpoint pathways
for DNA repair?
53
The DBF4 N-terminal conserved domain is not
required for viability
BRDF BRCT and DBF4 similarity
54
Loss of the BRCT-like domain causes defect in
response to DNA damage
Dbf4p
C
BRCT
M
704
1
350
Essential domain
Cdc7p-Dbf4p

• Promotes DNA repair
• Is required for firing late replication origins

BRCT domains are present in proteins involved in
the DNA damage response Protein interaction
domains - phospho-peptide binding
55
Mec1p and Rad53p phosphorylate Dbf4p following
exposure to HU
DNA damage
hrs in HU
0 1 2
Dbf4p
Mec1p (ATR)
Tel1p (ATM)
WT
Cdc7p
Ddc2 (ATRIP)
rad53-1
Chk1p
Rad53p (Chk2)
Dbf4p
Slows DNA replication
Cdc5p (Plk)
Dun1p
Securin
Anaphase
Late origins?
Crt1
Bub2p
Tem1p (GTPase)
DNA repair genes
Mitotic Exit
56
Telomeres occur at the ends of chromosomes
5
3

G-rich
Nucleosomal
Non-nucleosomal
Repeated DNA sequence
TG(1-3) budding yeast T2AG3 human cells
57
The End Problem
DNA polymerases require a template primer to
synthesis DNA No de novo DNA synthesis Polymeras
es only synthesize DNA in the 5 to 3
direction DNA primase can synthesize a short RNA
primer without a template, but RNAs are removed
during DNA synthesis because they are unstable
5
3
Lagging
RNA
Leading
5
3
58
The End Problem
DNA polymerases require a template primer to
synthesis DNA No de novo DNA synthesis Polymeras
es only synthesize DNA in the 5 to 3
direction DNA primase can synthesize a short RNA
primer without a template, but RNAs are removed
during DNA synthesis because they are unstable
5
3
RNA is removed leaving a gap
5
3
59
If not repaired, chromosomes would shorten over
time
5
3
Telomere erosion
60
Telomerase is a specialized polymerase that
maintains telomere length
5
3
61
Telomerase is a specialized polymerase that
maintains telomere length
5
3
Telomerase contains an RNA that serves as a
template for DNA synthesis
Telomerase is an RNA-directed DNA
polymerase Reverse Transcriptase
62
Telomerase consists of an RNA and a protein
component
Est2p is the catalytic subunit of telomerase and
belongs to the reverse transcriptase family
TLC1 is a 1.2kb RNA
Telomerase in a Ribonucleoprotein RNP
63
Telomerase synthesis at the ends of chromosomes
Vega et al. (2003) Nat. Rev. MCB 4948-959
64
Telomere associated proteins in human cells and
budding yeast
Blackburn (2001) Cell 106661-673
65
Telomere loops and folding
Vega et al. (2003) Nat. Rev. MCB 4948-959
66
Conservation of telomerase and associated proteins
Vega et al. (2003) Nat. Rev. MCB 4948-959
67
Telomere associated proteins in human cells and
budding yeast
Rap1p is a DNA binding protein that recognizes
telomeric DNA
Rap1p recruits the SIR complex
SIR complex recruits Ku
Blackburn (2001) Cell 106661-673
68
The ssDNA-binding protein Cdc13p recruits
telomerase to the ends
Cdc13p binds ssDNA and also interacts with
Est1p
Est1p is a subunit of telomerase that
binds to Est2
Est1p
Est2p interaction
Can bypass requirement for Est1p by making a
Cdc13p-Est1p fusion
Telomerase
Cdc13p
Blackburn (2001) Cell 106661-673
69
Ku tethers telomeres to the nuclear periphery in
budding yeast
In yeast and more complex eukaryotes, telomeres
are clustered together and interact with the
nuclear periphery
In the absence of Ku, telomeres are dispersed
throughout the nucleus and no longer show this
clustering arrangement
70
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir3-4
Rap1
Rap1
Rap1
Telomere
Nucleosomal DNA
Rap1p binds to telomeric DNA and recruits
Sir3p-Sir4p
Sir4p also recruits the Sir2p histone deacetylase
71
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir2p
Sir3-4
Rap1
Rap1
Rap1
Telomere
Nucleosomal DNA
Rap1p binds to telomeric DNA and recruits
Sir3p-Sir4p
Sir4p also recruits the Sir2p histone deacetylase
72
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir2p
Sir3-4
Rap1
Rap1
Rap1
Telomere
Nucleosomal DNA
Rap1p binds to telomeric DNA and recruits
Sir3p-Sir4p
Sir4p also recruits the Sir2p histone deacetylase
Sir3p-Sir4p bind to hypoacetylated histones H3
and H4
73
The Sir2p deacetylase is conserved
Yeast
Bacterial
Human
74
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir2-4
Sir2-4
Rap1
Rap1
Rap1
Telomere
Sir2-Sir3-Sir4 binding leads to deacetylation of
an adjacent nucleosome
75
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Rap1
Rap1
Rap1
Telomere
Sir2-Sir3-Sir4 binding leads to deacetylation of
an adjacent nucleosome . and results in the
spreading of the SIR complex along chromatin
76
Sir2-4 collaborate to form heterochromatin
adjacent to telomeres
Sir4p
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Rap1
Rap1
Rap1
Sir2-4
Telomere
Sir2-Sir3-Sir4 binding leads to deacetylation of
an adjacent nucleosome . and results in the
spreading of the SIR complex along chromatin
77
Some ARS elements are within the boundaries of
this heterochromatin
Sir4p
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Rap1
Rap1
Rap1
Sir2-4
Telomere
ARS
78
Position of ARS elements along chromosome III
Early origins
302 303 320
314
318
301
308
300
304
305
306
309
310
313
315
316
317
319
307
Chromosome III
lt 10
Inactive
lt 10
Late replicating
Telomeres
79
Loss of SIR complex advances replication timing
at telomeres
Sir4p
Ku
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
WT
Rap1
Rap1
Rap1
Sir2-4
Telomere
ARS
(late)
Ku
Sir4p
Rap1
Rap1
Rap1
sir3?
Telomere
ARS
(early)
80
Ku70? advances replication timing at sub-
telomeric ARSs
Sir4p
Ku
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
WT
Rap1
Rap1
Rap1
Sir2-4
Telomere
ARS
(late)
ARS501 (late)
81
Ku70? advances replication timing at sub-
telomeric ARSs
Sir4p
Ku
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
Sir2-4
WT
Rap1
Rap1
Rap1
Sir2-4
Telomere
ARS
(late)
ARS501 (late)
Sir4p
Rif1p
Rap1
Rap1
Rap1
ku70?
Telomere
ARS
(early)
ARS501 (early)
82
The centromere and kinetochore
Centromere
CdeII
CdeI
CdeIII
125bp
83
The centromere and kinetochore
Centromere
CdeII
CdeI
CdeIII
125bp
The Kinetochore is a proteinaceous structure
built upon the centromere for microtubule capture
and chromosome segregation
60 polypeptides
84
Linear models of centromere organization
The relatively simple centromere of
S. cerevisiae is located on an essential
125-bp region of DNA comprising three
functional DNA elements. cdeII is AT-rich and can
bind to the centromeric nucleosome Cse4/CENP-A
however, the primary determinant of
centromere location is cdeIII, which is bound by
CBF3 and essential for the localization of all
other kinetochore proteins. cdeI is bound by CBF1
and not essential, but its deletion results in
chromosome loss. In S. cerevisiae, homologues of
three human foundation kinetochore proteins,
Mif2/CENP-C, Mtw1/MIS12 and Nnf/CENP-H, exist as
part of larger multi- protein complexes an
arrangement that might also apply to higher
eukaryotes.
Trends Cell Biol. 2004 Jul14(7)359-68. Amor DJ,
Kalitsis P, Sumer H, Choo KH
85
Three dimensional view of a human
centromere/kinetochore
Trends Cell Biol. 2004 Jul14(7)359-68. Amor DJ,
Kalitsis P, Sumer H, Choo KH
86
The spindle checkpoint prevents mitosis
(anaphase) until all kinetochores attach
Cdc20p is a key mitotic regulator that is
kept inactive until all kinetochores have
attached to microtubules
Mad2p
Spindle Checkpoint genes
Mad2p-Cdc20p
MAD1, 2, 3 BUB1, 3 MPS1
(Inactive)
87
Spindle checkpoint prevents anaphase onset by
inhibiting cohesin degradation
Musacchio and Hardwick Nat Rev MCB (2002)