Prokaryotic DNA Replication

? Prokaryotic DNA Replication

DNA replication is perfomed by a multienzyme complex >1 MDa

DNA Nucleotides Replisome: DNA polymerases Helicase Primase SSBs DNA ligase Clamps (Topoisomerases)

1

Replication is semiconservative, accurrate and fast

Accuracy 1 error in 1 billion bases Speed 500 nt/s in bacteria 50 nt/s in mammals

Each original strand functions as template for DNA synthesis

2

After each replication cycle, DNA is doubled

DNA is synthesized in 5?to 3?direction

3

Polymerisation in detail

(dNMP)n + dNTP DNA

(dNTP)n+1 + PPi 2 Pi

Complementary basepairing and matching hydrogen bonds is required

Incorrect basepairing

4

DNA is synthesized by DNA polymerase

DNA polymerase III is a protein complex

Subunit

function

not known 3' exonuclease polymerase clamp dimerisation

clamp loader

5

E. coli contains multiple DNA polymerases

DNA pol I

DNA pol II

DNA pol III

Number/cell

400

100

10

Speed (nt/s)

16-20

2-5

250-1000

3?exonuclease Yes

Yes

No

5?exonuclease Yes

No

No

Processivity

3-200

10 000

500 000

Role

DNA repair

DNA repair

Replication

RNA primer

removal

DNA polymerase I

Found by Arthur Kornberg, mid 1950's

Three enzymatic activities:

?

Polymerase activity

?

3' to 5' exonuclease activity

?

5' to 3' exonuclease activity

Klenow enzyme is lacking one subunit responsible for the 5' to 3' exonuclease activity

6

DNA polymerase requires

1. A free 3'-OH group supplied by RNA Primer for start of polymerisation

2. Mg2+ ions for activity in active site 3. A template to copy

DNA replication initate at origin of replication

Bacterial chromosome doubles in 40 min

7

DNA replication is bidirectional

The replication origin OriC in E.coli

245 base pairs AT-rich Initiation proteins bind to 9 bp consensus sequence

8

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