DNAReplication and Repair

1
DNA-Replication and Repair in Prokaryotes
2
E. coli
3
Meselson-Stahl Semiconservative Replication
4
One Ori Bidirectional DNA Replication
5
H3-Labelling Origin (Ori) Direction of
synthesis 5to 3
Pro/Eukaryoten
6
The Replication Fork How can both strands be
synthesized simultaneously?
Initiation Elongation Termination
7
Primer
?
8
DNA Polymerase 1 (103 kDa protein)
Elongation
Substrate recognition
RNA
9
DNA-Polymerase 1 substrates Geometry! Polymerisat
ion!
10
Different geometry no substrates! 3-Exonuclease
11
Error correction by the 3---5Exo. Proofreading!

And 5----3Exo
Is DNA polymerase 1 essential?
12
DNA-Polymerase-103kDa- has 3 modules Klenow
Fragment (ca.67 kDa) polymerase Proofreading
3-Exo 5Exo
13
Role of DNA polymerase 1 5-Exo removes RNA
primers
Nick-Translation
14
Primer
?
15
DNA Polymerase III
16
3D-structure 2-b subunits DNA
17
3D-structure 2-b subunits DNA
donut-shape
18
DNA polymerase 1 5-exonuclease activity Ligation
19
DNA-Ligase
Viren/ Menschen
E. coli
20
Initiation site of replication The E. coli origin
of replication oriC (245bp)

• Initiation
• Elongation
• Termination

21
Initiation
22
Model for initiation at oriC
Regulation? ATP Methylation at GATC (x11)
23
Elongation and synthesis Okazaki fragments
24
Single assymetric DNA polymerase III
25
Termination-Trap (no exit)
TusTermination utilization subs.
26
DNA separation (catenanes)
(type Top II)
27
E. coli
28
DNA Repair
29
Ames Test for Carcinogens (Salmonella typhimurium)
Spontaneous mutants on His-free medium
30
Fehlpaarungsreparatur
31
DNA Metylation and Mismatch repair
32
Model for Methyl-directed mismatch repair
DNA Abbau DNA Synthese
DNA-Helikase Exonukleasen
33
Mismatch repair
5-Exo
3-Exo
ca. 1000 bp
34

• Nicht-enzymatische Veränderungen von Nucleotiden
  und
• Nucleinsäuren
• Desaminierung
• Depurinierung
• Cyclobutan-Thymindimer
• Chemische Modifikation

35
Desaminierung
36
Depurinierung
ß-Furanose In die Aldehydform
37
Base Excision repair AP-Site Cytosin
deamination Uracil-glycosylase DNA-glycosylase
38
Nucleotide Excision Repair
Human
E. coli
39
UV-Licht induziert die Bildung von
Cyclobutan-T-T Dimere
40
(No Transcript)
41
Direct Repair Photon absorbtion blue
light (300-500 nm) by MTHF-(photoantenna)
PhotolyaseThymine dimers 54kDa, Chromophores
FADH2, 5, 10-Methenyltetrahydrofolylglutamat
42
Desaminierung
43
Alkylierende Agentien
SAM
44
Dimethylsulfate
Keine Paarung mit C
45
Alkylating agents
46
Mutation by Alkylation
47
Direct Repair by Metyltransferase
Transcriptional activator!
48
DNA damage and DNA Rplication
DNA Recombination