Transcript No Slide Title

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.
I
Wait a minute!
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.
200
polA + (wild type)
II
0.4M
III
0.2M
100
+NEM
200
polA- (Cairns)
II
0.4M
III
0.2M
100
I
20
30
Fractions
40
Phosphate (M)
John Cairns mutated the
gene for DNA
polymerase, polA, and
the bacteria grew just
fine!
3H Thymidine incorporation (pmol)
600
Sub kDa
unit
Gene


130
27.5

10
dnaE
|
dnaQ
| POL III CORE
(mutD)
|

71
dnaX


'


47.5
35
33
15
12
dnaX

40.6
dnaN
Subassembly
|
|
|  COMPLEX
|
|
 CLAMP
DNA POLYMERASE III
5'-3' polymerase
5'-3'
exonuclease
3'-5'
exonuclease
ATP dependent
clamploader
processivity
factor
Sub Gene
unit
Bacterial
Function
Eukaryotic

dnaE
|
DNA POL 


dnaQ
| POL III
(mutD) CORE
|
5'-3'
polymerase
3'-5'
exonuclease
5'-3'
exonuclease

dnaX


'
dnaX



dnaN
|
|
|
COMPLEX
|
|
 CLAMP
ATP
dependent
clamploader
processivity
factor
DNA POL 
Fen1
RF-C
PCNA
CONSERVATION FROM PROKARY OTES TO
EUKARYOTES
Which polymerase is processive?
P
POL
dNTP
Challenge with vast excess of cold
primer-template
Gel electrophoresis of products
- +
Challenge +
POL-X
POL-Y
POLIII,  subunit
PCNA
Clamp loaders hydrolyze ATP to load clamp
Clamp-loader
ATP
ATP
Clamp
How does one prove
that the clamp ring
is opened during
loading?
ATP
ADP
+ PPi
3‘OH
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 relax DNA by changing the DNA
linking number
* 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 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 of DNA in gate
segment is covalently
linked to the OH of
tyrosine in the active
site of topo.
Cycle of topoisomerase activity inferred from structure
1
2
4
3
How would you test that the subunits have to open at the lower end to release the T segment?