A,

DNA
DNA Replication, Repair, and Recombination


, , ,
. DNA

 DNA
DNA Replication
To DNA .
DNA =
( / )
. DNA
1.

(base pairing) C G,

A T.

DNA

-
J. Watson F. Crick, 1953

3
2. 3. DNA pol

2. DNA
DNA
()
() . DNA


.

3. DNA
(
<1:1010, ),
~1000 n/sec
(E.coli DNA 4.8 106 bp, <40min), ?????

(processive vs distributive)

4
,, DNA

DNA
DNA polymerase

II

III

polymerase

5 exonuclease

exonuclease

>10

. /sec

20

40

250-1000

3-200

1500

>500.000

mol/ cell

400

pol A

pol B

(proof reading)

10-20 Why ?
pol C

H
DNA

(Mg++)

.
,
PPj

 DNA pol III

2 core complexes (, , ),

2 , complexes (2 ,2)
2: (sliding clamp)
1 complex,
,,,,

,
(replisome)

DNA
3-5
.

4. DNA

H PcrA

5.

:
T : DNA
(DNA )

8
Ori C

6. DNA
,

E. coli genome

--- ,

5
3

5
3
5

3
3

5
3

10
.

. DNA
1. . Coli
oriC (origin of Chromosomal replication).
To oriC 4
DnaA

11
DnaA-B- SSB

2. -.
DnaA, DnaB () SSB (single strand binding)

DnaA oriC

(/)

DnaB

DnaB

-
SSB

SSB

SSB SSB
SSB
DNA

12

primase

3.
RNA (primer)
( RNA ) DNA
H DNA
,
.

(primase)
-
(~10n)
RNA

To RNA
DNA DNA .
5->3 DNA .

3
5

5
3

13
okazaki

 DNA (, leading
strand), ( / , lagging strand)
( Okazaki)
5

DNA pol
DNA

5 -> 3

3
3

5 3

5
5

Okazaki

DNA , o
RNA
5->3 -
DNA .
(Nick translation)

5
3

Okazaki

3
3

5
3

14
ligase

 DNA (ligase) 3- DNA

53 , .
Okazaki

15

 DNA

Lys
O

1. Ligase (E) + ATP PPi

E -AMP

NH

O P
O

Ligase (E)

E -AMP

OH OH
O
O
O P
O

2.

E-AMP + P-5-DNA E +

AMP--DNA

3. DNA-3'--OH +

O P
O

P O

5'-DNA

O5-5

AMP--DNA

OH OH

O
O

P O

5'-DNA

O-

AMP--DNA

DNA-3- O

P O

5'-DNA + AMP-OH

O-

OH OH

16

 DNA

DNA DNA . ?
5
3

5
3
5

3
3

5
3

DNA.
17
DNA pol

/ DNA III

/ Processivity

Sliding clamp

loader

Polymerase
3'-5' exonuclease

Polymerase

18

 -. -
,

(PCNA, proliferating cell nuclear antigen)

19

 DNA

(DNA
, ), SSB
.
-

.
!!!

DNA RNA primers, DNA

Okazaki ,
RNA DNA .
DNA

DNA, DNA
.
20

 DNA

DNA ( E. Coli 4.8 106 bp vs Human 6 109 bp),

, .
DNA , , , , , .
(: repair, : mitochondrial)

S .
(licensing factors)
.
()
!!

21
ARS cloning

 (ARS: Autonomously replicating sequence)

DNA

DNA

His+

+
DNA-ARS

His

22
Acyclovir

 (Gertrude Elion)
DNA

(HSV)

()
-

HIV

thymidine

azidothymidine
(AZT)

23
t


(telomerase)

5
5

3
3

5
3

,
() !!!

24

H 3
5
5
3
3
5

3
3
5

3
3
5

H RNA
RNA ??

25

 DNA (DNA Recombination)

H DNA

,

DNA
Holliday and
Whitehouse (1964).
26

Heteroduplex DNA
M
,
Nick,

27

Holliday structure

28


1. (deletion)
2. (insertion)
3. (substitution) :
(transition) Pu->Pu, Py->Py
(transversion) Pu->Py, Py->Pu

DNA
`
:
1. ( )
DNA
2.
3. (, UV, X-rays)
Oi !!!

29
HNO2

1. X DNA

NH2
N

NH

CH3

HN

NH

C
NH

e
NH2

O
N
NH

NH2

NH

N
O

O
HN

NH

-> C
-To 2
-: H
,

CH3

NH

6eG
G -> A
-
6 , 3
(3Me-A)
30

IIIIIIIIIIII

CH3-G

IIIIIIIIIIII

IIIIIIIIIIII

IIIIIIIIIIII

CH3-G

IIIIIIIIIIII

IIIII IIIIII

IIIIIIIIIIII

Me

IIIIII IIIII

IIIIIIIIIIII

G -> A

CH3-G

IIIIIIIIIIII

6eG

NH

IIIIIIIIIIII

IIIIIIIIIIII

HN

CH3

Oups !!!

C to U

31

2. in vivo
NH2

O
HN

NH

H2N

NH

O
H3C

NH

NH

U
NH

NH2

NH

N
NH

U
U DNA

32

3. UV ,

UV

33

4.
: A
/ .
.. 5-Br-

NH2

O
Br

BrU

> C=O

NH

NH

H 5-Br-

NH

C-OH

OH
Br

HN

N
BrU*

NH

H2N

G
N

A ->G

NH

34

( CY450)

35
Ames

 Ames



,


His-

His-

His+

His+
,

-

36

 DNA
1. (direct repair)
2.
3.

1. :
-- : DNA
DNA
(E.coli, yeast)
-- - 6MeG e o

37
C->U

2.

NH2

O
HN

NH

H2N

G
N

NH

- DNA-pol

U
NH

/ , DNA
!!

NH2

NH
O

(
)

NH

38

3.
--

-8, +4


(excinuclease)
DNA .

(xeroderma pigmentosum)



( )
39
Lynch

---
(mismatch repair)



( Lynch 1:200!!!)
M hMSH1 hMSH2


hMSH1 hMSH2
MutS, MutL
E. coli

40
huntigtine

Huntigtons disease
CAG (polyQ)

---- CAG CAG CAG CAG ------ CAG CAG

--- GTC GTC

---- CAG CAGCAG CAG ---CAG CAG CAG CAG ---

/ (nticipation):

,

6-30 Q 35-80Q
41


Stryer . 876-877
1, 3, 6, 7, 11, 15.
Lehninger K. 26
2, 5, 6, 9.

42

43
ARS cloning

 DNA

44

 DNA

B-DNA: , 10 bp
A-DNA: 11 bp , RNA/DNA and RNA/RNA.
Z-DNA: .
: Pu- Py- Py
45

 ( )

46

5.

: ,
, DNA
: DNA,
DNA

(DNA )

47
Ori C


: A
( )

NH2
N

-2

NH

10-4

O
HN
O

*
NH

NH

NH
N

CH3

NH2
N

NH
O

C
NH

-> C
48
Br-U