斯坦福大学分子生物学讲义(英文)

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
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.
* 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.
PCNA
Clamp loaders hydrolyze ATP to load clamp
Clamp-loader ATP
ATP
How does one prove that the clamp ring is opened during loading?
Clamp
ATP
ADP + PPi
3慜H
Structure of a DNA polymerase (gp43 from phage RB69)
* 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.
40.6 dnaN CLAMP
processivity
factor
DNA POLYMERASE III
Sub Gene Bacterial unit
Function
Eukaryotic
dnaE |
dnaQ | POL III
(mutD) CORE
|
5'-3' polyme rase 3'-5' exonuc leas e 5'-3' exonuc leas e
DNA POL DNA POL Fen1
dnaX
dnaX |
|
ATP
'
|
dependent RF-C
COMPLEX clamploader
|
|
dnaN CLAMP processivity PCNA factor
CONSERVATION FROM PROKARYOTES TO EUKARYOTES
3H Thymidine incorporation (pmol) Phosphate (M)
I
600 200
III
100
+ NEM
polA + (wild type) 0.4M II
0.2M
200
polA- (Cairns) II
0.4M
III
100
0.2M
I
20
30
40
Fractions
Sub kDa Gene Subassembly unit
130 dnaE |
5'-3' polwenku.baidu.commerase
27.5 dnaQ | POL III CORE 5'-3'
(mutD)
exonuc leas e
10
|
3'-5'
exonuc leas e
71 dnaX
47.5 dnaX |
35
|
' 33
| COMPLEX
15
|
12
|
ATP dependent clampl oade r
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.
Side view: Polymerase active site
Top view with template-primer: Polymerase site And proofreading site
Topoisomerases relax DNA by changing the DNA linking number