Looping the
lagging
strand to
make both
polymerases
move in the
same
direction
The discovery of DNA polymerase.
Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain
of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen.
The enzymatic activity was unusual:
1) Needed a template which dictates what nucleotide was added,substrate was directing enzymatic activity
2) Needed a primer annealed to the template.
Wait a minute!
John Cairns mutated the
gene for DNA
polymerase,polA,and
the bacteria grew just
fine!
Either the polymerase
hypothesis was all
wrong,…… or there were
other DNA polymerases
in E,coli
that carried out DNA
synthesis in the polA
strains.
0, 2 M
0, 4 M
100
200
20 30
40
Frac t ions
pol A- ( C ai r n s)
I
III
II
100
200
600
3
H
T
h
y
m
i
d
i
n
e
i
n
c
o
r
p
o
r
a
t
i
o
n
(
p
m
o
l
)
0, 2 M
0, 4 M
P
h
o
s
p
h
a
t
e
(
M
)
pol A + ( wild ty pe)
+ N E M
III
II
I
S ub
un i t
k D a G e n e S ub a ss e m b l y
? 1 3 0 d n a E | 5 '- 3 ' po l y m e ra se
? 2 7, 5 d n a Q
( m ut D )
| P OL I I I C O R E 5 '- 3 '
e x o n u c l e a s e
? 1 0 | 3 '- 5 '
e x o n u c l e a s e
? 7 1 d n a X
? 4 7, 5 d n a X |
? 3 5 | A T P d e p e n d e nt
? ' 3 3 | ? CO MP L E X cl a m p l o a d e r
? 1 5 |
? 1 2 |
? 4 0, 6 d n a N ? C L A M P p r oc e ss i v i t y
fa c to r
D N A POL Y ME R A S E II I
S ub
un i t
G e n e B a c t er i a l Fu n c t i o n E uk ar y o t i c
? d n a E |
5 '- 3 '
po l y m e ra se
D N A P OL ?
? d n a Q
( m ut D )
| P OL I I I
C O RE
3 '- 5 '
e x o n u c l e a s e
D N A P OL ?
? |
5 '- 3 '
e x o n u c l e a s e
Fe n 1
? d n a X
? d n a X |
? |
A T P
? ' | ?
C O M P L E X
de p e n d e nt
cl a m p l o a d e r
R F- C
? |
? |
? d n a N
? C L A M P p r oc e ss i v i t y
fa c to r
P C N A
CO N SE R VA T I O N F R O M P RO K A R Y O TE S T O
E U K A R Y OT E S
P
P O L d N T P
C hall en ge wi th vast e xcess of co ld
prim e r -temp late
Gel electr o pho resi s o f pro d ucts
C h a l l e n g e - + - +
POL- X POL- Y
Which polymerase is processive?
POLIII,? subunit PCNA
A T P
A T P
3 慜 H
A T P
A D P
+ PP i
C l a m p
C l a m p - l o a d e r
Clamp loaders hydrolyze ATP to load clamp
How does one prove
that the clamp ring
is opened during
loading?
Structure of a DNA polymerase (gp43 from phage RB69)
Side view:
Polymerase active site
Top view with
template-primer:
Polymerase site
And
proofreading site
* Topoisomerases II change the linking number in steps of 2 by
passing both strands of double-stranded DNA through a break,
* Eukaryotic topoisomerases isolated to date only relax
supercoiled DNA,while prokaryotic topoisomerases (gyrases)
can,given ATP,add supercoils.
* TopoII releases catenated daughter molecules at the end of
replication,Inhibitors like etoposide are used in chemotherapy.
Topoisomerases relax DNA by changing the DNA
linking number
* Topoisomerases I change the linking number in steps of 1,They pass a
single DNA strand through a nick.Topoisomerase I is a protein of the
metaphase chromosome scaffold,
* In interphase,topoisomerase is bound to the nuclear matrix,
* The DNA replication machinery also appears bound to the matrix,
* Inhibitor (camptothecin) also used in chemotherapy.
Topoisomerase action can be divided into three steps,
nicking (1),strand passage (2); resealing (3).
5? end o f D NA i n ga t e
seg me nt is co val entl y
li nk ed t o the OH of
tyros ine i n the ac t ive
sit e o f topo,
1
3
2
4
Cycle of topoisomerase activity inferred from structure
How would you test that the subunits have to open at the lower end to release the T segment?