Download DNArepl2

Characteristics used to define Origins of Replication
* The position on the DNA at which replication start points are
found. (4 methods discussed)
* A DNA sequence that when added to a non-replicating DNA
causes it to replicate.
* A DNA sequence whose mutation abolishes replication.
* A DNA sequence that in vitro is the binding target for enzyme
complexes known to function in initiation of DNA replication.
1) In synchronously replicating cells, density transfer experiments can
reveal which sequences replicate earliest. What is the direction of fork
movement here?
% of probe in HL DNA
Genes on a
chromosome
A
B
C
100
C
B
0
Time
A
TR50 Calculation for human chromosomal probes
on genome tiling arrays (ENCODE project)
TR50 - Time at which 50% of the locus is replicated
In the example below,
‘probe A’ has a TR50 of 1.25hr (80% at 2hr, 0% at 0hr)
‘probe B’ has a TR50 of 6.33hr (100% at 8hr, 40% at 6hr)
Example:
TR50
100
Time Point
Probe A
Probe B
0-2
.80
.10
2-4
.10
.10
4-6
.10
.20
6-8
0
.60
% cumulative replication
90
80
Gene
Probe A
Gene
Probe B
70
60
50
40
30
20
10
0
0
2
4
Hr of S phase
6
8
Predicted origins, UCSC genome browser,
ENCODE: UVa DNA replication tracks
Neerja Karnani
Chris Taylor
Ankit Malhotra
2) Electron microscopy of bubbles after restriction
enzyme digestion
BamHI
BamHI
EcoRI
EcoRI
3) Hybridizing lagging strand nascent DNA to strand-specific fragments
shows site of switch from leading to lagging strand synthesis
Upper strand A
fragments
Lower strand
fragments
E
B
F
C
D
G
H
Nascent DNA used to probe DNA fragments:
A
B
C
D
E
F
G
H
Autorad after hybridizing
nascent strands
A
B C D
E
F
G
H
DNA spotted
on membrane
3b) Hybridizing of leading strand nascent DNA shows sites near
origins that are enriched in nascent strand
Nascent strand abundance
E
F
Probes
A
B
C
G
D
H
Okazaki fragments: <500 bases long
Leading strand nascent strands: > 500 bases long
Leading strand nascent strands near origins < 2000 bases long
Nascent DNA 500-2000 base long used to
probe fragments along chromosome
E F A
E F A
B C D
B C D
G H
G H
Nascent strand peak at origin
DNA spotted on array
4) 2D Gel electrophoresis of replication intermediates followed by
hybridization with a DNA fragment reveals whether a replication
bubble originates in the fragment
Dimension 1: separates by size; dimension 2: separates by shape
Sequence that when added to a non-replicating DNA causes it to
replicate; this approach was used to identify
Autonomously Replicating Sequences (ARS) in yeast.
Sequence that when mutated causes a replicating DNA to
fail to replicate; this approach was used to identify
essential elements in an (ARS) in yeast
Yeast containing
Leu2 on a plasmid
Grow under
non-selective
conditions
Plate yeast on Leucine minus plates
to estimate rate of plasmid loss
Linker scanning mutations in yeast ARS1
Examples of
mutants
% URA+ colonies
WT-ARS1
B3
B2
B1
A
OriC in E. coli chromosomal DNA
Initiation of DNA replication in E. coli
Nature Structural
& Molecular Biology –
13, 676 - 683 (2006)
Berger JM
DnaA and oriC independent replication initiation in E. coli
from hairpins, D-loops and R loops
Two primosomes for E. coli chromosomal replication
Site of primosome assembly
oriC, A-site
n'-pas, D-loop, R-loop
Recognition
DnaA protein
PriA protein
Auxilliary proteins for
loading helicase
(HU for oriC)
PriB
PriC
DnaT
Helicase
DnaB/DnaC
Priming
Primase
SV40: a polyomavirus that is very useful for
studying eukaryotic replication enzymes
Early genes : proteins essential for viral DNA replication
T antigen binding sites cluster around the ori
Properties of SV40 T antigen
*
*
*
*
*
*
98% nuclear: NLS
origin-specific DNA binding
DNA independent ATPase
ATP dependent DNA helicase
Binds to DNA polymerases, AP2, p53, Rb
Undergoes phosphorylation etc.
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
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