Download DNArepl3

SV40 ori
Li & Kelly
SV40 DNA replication
In vitro.
PNAS (1984) 81:6973
COS cells
(infected with SV40)
(expresses T Ag)
+ plasmid + dNTP + rNTP
+ 32PdATP
37o C, 60 min
Analyze products by electrophoresis
and autoradiography
SV40 +
ori +
What else could they do
to validate their system?
Fractionation to homogeneity
COS cells
(infected with SV40)
(expresses T Ag)
Human HeLa
or 293 cells
T Antigen
from
cell lysate + baculovirus
expression
system
PC
0.2 M KCl
0.66 M KCl
(RF-C, pol  + pol )
QS
0.2 M KCl
0.4 M KCl
(PCNA)
ssDNA
0.6 M KCl
1 M KCl
RPA
ELONGATION FACTORS ARE CONSERVED
Prokaryotic
Function
Eukaryotic
SSB
ss DNA coating
RPA
PRIMASE
RNA primer synth.
Primase subunit
of DNA POL ,
5'-3' polymerase
POL III CORE 3'-5' exonuclease
5'-3' exonuclease
DNA POL ,
DNA POL 
DNA POL 
Fen1
 COMPLEX
ATP dependent
clamploader
RF-C
 CLAMP
processivity factor
PCNA
LIGASE
seal nicks
Lig1
DnaB
Helicase
?
Sub Gene
unit
Bacterial
Function
Eukaryotic

dnaE
|
DNA POL 

dnaQ
(mutD)
| POL III
CORE
|
5'-3'
polymerase
3'-5'
exonuclease
5'-3'
exonuclease
|
|
|
COMPLEX
|
|
ATP
dependent
clamploader


dnaX


'
dnaX



dnaN
 CLAMP
processivity
factor
DNA POL 
Fen1
RF-C
PCNA
CONSERVATION FROM PROKARYOTES TO
EUKARYOTES
RF-C
RF-C
RF-C is a five-subunit complex
All subunits are related in sequence and have ATP binding motifs
ATP hydrolysis by RF-C is associated with the loading of PCNA
RF-C is the functional homolog of the clamp-loader  complex


Polymerase switching occurs even on lagging strands;
pol  does most of DNA synthesis
Dna2 endonuclease is also necessary for this step
How do you think the PCNA is removed after ligation?
PCNA interacts with RF-C, pol , Fen1, DNA ligase,
CAF1 and MCMT
Several of these have a common motif
used in the interaction: Q-X-X-L/I/M-X-X-FF/Y
p21/CIP1/WAF1, a protein induced by the
tumor suppressor p53 uses the same motif
to interact with PCNA
What effect is p21 expected to
have on DNA replication/repair?
FEN1 interacts with PCNA
ELONGATION FACTORS ARE CONSERVED
Prokaryotic
Function
Eukaryotic
SSB
ss DNA coating
RPA
PRIMASE
RNA primer synth.
Primase subunit
of DNA POL ,
5'-3' polymerase
POL III CORE 3'-5' exonuclease
5'-3' exonuclease
DNA POL ,
DNA POL 
DNA POL 
Fen1
 COMPLEX
ATP dependent
clamploader
RF-C
 CLAMP
processivity factor
PCNA
LIGASE
seal nicks
Lig1
DnaB
Helicase
?
DNA Helicase
15mer
3‘
5‘
P
5‘
* Helicase binds ss DNA
* Hydrolyzes ATP to move along
ssDNA and peel of substrate DNA
* Can move 5‘ to 3’ or 3‘to 5’ or
in both directions on ssDNA
and is classified as such
30mer
3‘
P
5‘
3‘
te
P
a
T
r
A
st
+
b
e
e
te
u
s
s
a
s
a
tr
ca
c
d
i
i
s
l
l
b
e
ile
e
u
h
h
o
S
+
+
B
* Dimers or hexamers
30mer
15mer
Helicases are ATP driven molecular motors
Conserved helicase motifs:
Includes the following for ATP
binding and hydrolysis
I = Walker A motif GxGxGKT
II = Walker B motif FFFDEad
Binds ATP
Tight conformation
Hydrolyzes ATP
Relaxed conformation
Mutation in a yeast gene that causes a failure in
MiniChromosome Maintenance (MCM genes)
Yeast containing
Leu2 on a plasmid
Grow under
non-selective
conditions
Plate yeast on Leucine minus plates
to estimate rate of plasmid loss
Form hexamer
Associates with
MCM2-7
Newbies of
unknown function
MCM8
MCM9
MCM homolog from
M. thermoautotrophicum
(an archaebacterium)
has helicase activity.
What direction?
Mt MCM forms
double hexamers
T antigen helicase domain:
XS Chen, Nature, 2003;423:512-8
MtMCM-N terminal (non-helicase) domain:
XS Chen, Nature Str. Biol. 2003;10:160-7
Model of replicative double-hexameric helicase
action : XS Chen, Nature, 2003;423:512-8
Formation of pre-Replicative complex
Annual Review of Biochemistry
2002. Bell and Dutta
Replication complexes during the cell cycle
CDC6+
Cdt1
ORC
Replication
Machinery
late G1
CDC6 +
Cdt1
MCM
S
MCM
“Pre-Replicative Complex”
early G1
G2
M
ORC
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as
being the eukaryotic DNA replication initiator protein.
Subunits are named according to their size, with ORC1 being the
largest and ORC6 being the smallest subunit.
Yeast ORC specifically binds to replication origins in an ATP
dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the prereplicative complexes that co-operates with Cdt1 to load MCM2-7
Proteolyzed in yeasts or exported out of the nucleus in mammalian
cells at the G1- S transition.
Overexpression of Cdc6 in yeast causes multiple rounds of DNA
replication without intervening mitosis, making it a critical regulator
of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem
to function together in a large multi-subunit protein complex.
MCM 2-7 is most likely the replicative DNA helicase.
Origins are multi-partite
Transcription
Factors
Auxiliary
elements
Origin recognition
proteins
A/T
ORE
Core origin
Leading str Lagging str
Lagging str
Leading str
Transcription
Factors
DUE
Auxiliary
elements
The Structure of ARS1
(S. cerevisiae)
ORC
3
6
ABF1
B3
B2
5
22
1
4 1
ACS
B1
“Post-RC” protection
“Pre-RC” protection
4
ORC uses different strategies for binding DNA
in different species
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as
being the eukaryotic DNA replication initiator protein.
Subunits are named according to their size, with ORC1 being the
largest and ORC6 being the smallest subunit.
Yeast ORC specifically binds to replication origins in an ATP
dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the prereplicative complexes that co-operates with Cdt1 to load MCM2-7
Proteolyzed in yeasts or exported out of the nucleus in mammalian
cells at the G1- S transition.
Overexpression of Cdc6 in yeast causes multiple rounds of DNA
replication without intervening mitosis, making it a critical regulator
of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem
to function together in a large multi-subunit protein complex.
MCM 2-7 is most likely the replicative DNA helicase.
The Xenopus Cdc6 Protein Is Essential for the Initiation of a
Single Round of DNA Replication in Cell-Free Extracts Made
from Xenopus egg extracts
Only elongation required on
single-stranded DNA substrate
Replication initiation required on
double-stranded DNA substrate
32P labeled
DNA
replication
product
Rxn Time:
0-30 min
30-60 min
60-90 min
+
+
+
+
+
+
+
+
+
+
+
+
Orc is required
to load CDC6;
CDC6 is required
to load MCM
Immunostaining for CDC6 can pick out
proliferating cells in Pap smears
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as
being the eukaryotic DNA replication initiator protein.
Subunits are named according to their size, with ORC1 being the
largest and ORC6 being the smallest subunit.
Yeast ORC specifically binds to replication origins in an ATP
dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the prereplicative complexes that co-operates with Cdt1 to load MCM2-7
Proteolyzed in yeasts or exported out of the nucleus in mammalian
cells at the G1- S transition.
Overexpression of Cdc6 in yeast causes multiple rounds of DNA
replication without intervening mitosis, making it a critical regulator
of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem
to function together in a large multi-subunit protein complex.
MCM 2-7 is most likely the replicative DNA helicase.
Mutation in a yeast gene that causes a failure in
MiniChromosome Maintenance (MCM genes)
Yeast containing
Leu2 on a plasmid
Grow under
non-selective
conditions
Plate yeast on Leucine minus plates
to estimate rate of plasmid loss
Form hexamer
Associates with
MCM2-7
Newbies of
unknown function
MCM8
MCM9
Fluorescence analysis and cell sorting (FACS)
DNA in cells stained with propidium iodide.
Intensity of staining in each cell is proportional to amount of DNA in the cell.
Propidium iodide staining
MCM
Chromatin
immunoprecipitation
(ChIP) tests whether a
given DNA sequence
is bound by a protein
in vivo
Cross-link protein-DNA
I.P. with anti MCM antibody
anti-MCM Ab
Reverse cross-link
Purify DNA in I.P.
PCR to detect if your
sequence was I.P.ed
Role of Pre-RC in mammalian cells
ORC :
CDC6/Cdc18 :
Cdt1:
MCM2-7 :
Human ORC
Wild type cells
D/- cells
ORC4
Quintana, 1997, 1998
Pinto/Quintana, 1999
Thome 2000
Dhar, 2000, 2001
Mammalian cells can survive with 0.1x the
normal ORC2 level : WT vs D/- cells
Cell proliferation of ORC2 +/+, +/- and D/cells measured by MTT assay
Replication from an origin in Epstein-Barr virus:
a plasmid expressing EBNA1 and containing oriP will
replicate and be maintained as an episome in
mammalian cells
EBNA1 protein
oriP
HygromycinResistance
gene
Establishment of drug-resistant colonies
after transfection of EBV-based plasmid
DpnI-resistance assays episome replication in mammalian cells
m DpnI
X
m DpnI
DpnI
X
m
DpnI
DpnI
X
m
m
m
DpnI
X
m
m
Bacterial plasmid
Methylated
DpnI susceptible
m
DpnI
DpnI
X
Replicated once in
human cells
Hemi-methylated
DpnI resistant
DpnI
X
Replicated twice in
human cells
Unmethylated
DpnI resistant
1)EBV-based plasmid replication is decreased in D/- cells.
2) Rescued by plasmid expressing ORC2D
+ +
Some cancers have an Achilles Heel
Viral episomes that carry viral oncogenes
Amplicons of cellular oncogenes, or of drug resistance genes
Normal
Cancer