DNA Relication in Prokaryotes

（

3

）

Structure of DNA polymerase

Ⅲ

Pol

Ⅲ

holoenzyme——asymmetric dimer

leading strand synthesis lagging strand synthesis

















’

_

















Elongation speed ： 1000nt/min



Components of Pol III enzyme:

enzyme with

16

polypeptide

involving

10 subunits

1. Core enzyme:

(i

(ii) epsilon subunit: 3’-5’ exonuclease activity-proofreading function

(iii) theta subunit: Role is not yet clear

2. Beta (Processity component) beta clamp

:

3

. R-complex or Clamp loader or Match maker:

4

. Tau subunit

-

Clamp loading mechanism.

Leu F P , O'Donnell M J. Biol. Chem. 2001;276:47185-47194

1. Initiation of DNA Replication

①

Structure of the

oriC

245bp

7 repeating sequence ：

9mers ， 4

13mers ， 3 i ） Length ：

ii ） Composition ：

AT rich sequence-melting of DNA; Formation of

Pre-priming complex formation



_{Dna A}

_{DnaA-ATP is a positive modulator of initiation Of}

DNA replication

DnaA HU IHF

DNA gyrase

_{The first step in the formation of pre-priming complex is the}

binding of DnaA protein with 9 mer

_{The Dna A binding is cooperative process.The DnaA molecules go}

on binding till 20-40 molecules form a core or barrel of DnaA protein

_{HU (Histone like protein) and IHF (integration host factor) assist in}

binding of DNA around DnaA proteins

_{The nutshell effect of DnaA binding is to exert tortional stress that}

results in melting at 13 mer region

Pre-priming complex formation



_{Dna B}

_{It possess helicase activity and called DnaB helicase} _{Six molecules of DnaB form a hexamer.}

_{DnaB alone can not bind to single stranded DNA.}

Instead, it is escorted by DnaC which also called “match-maker”

_{DnaB-DnaC complex binds to single stranded region}

on DNA

_{The function of DnaB helicase is to assist in}

Pre-priming complex formation



_DnaC



_{Escorts DnaB on single stranded DNA template}



_{Thus, pre-priming reaction begins with}

binding of DnaA at OriC , and ultimately

results in binding of DnaB at the two

②

Proteins initiate unwinding

DnaA:

i ） Helicases

Break hydrogen bonds.

ii ） Single-stranded binding proteins （ SSBs ）

Stablize the open confirmation.

DnaB/DnaC:

SSBPs

（

）

stablize single strand DNA

p

SSBP

21-19

Priming in

E. coli

•

Primosome refers to collection of proteins

needed to make primers for a given

replicating DNA

•

Primer synthesis in

E. coli

requires a

primosome composed:

–

_{DNA B helicase}

–

Primase, DnaG

•

Primosome assembly at the origin of

21-21

Priming in

E. coli

•

Primosome refers to collection of proteins

needed to make primers for a given

replicating DNA

•

Primer synthesis in

E. coli

requires a

primosome composed:

–

_{DNA helicase}

–

DnaB

–

Primase, DnaG

•

Primosome assembly at the origin of

21-22

Origin of Replication in

E. coli

Primosome assembly at

ori

C occurs as

follows:

–

_{DnaA binds to}

_ori

_{C at sites called}

_dnaA

_boxes

and cooperates with RNA polymerase and HU

protein in melting a DNA region adjacent to

leftmost

dnaA

box

–

DnaB binds to the open complex and facilitates

binding of primase to complete the primosome

–

Primosome remains with replisome, repeatedly

primes Okazaki fragment synthesis on lagging

strand

–

DnaB has a helicase activity that unwinds DNA

•

_{The direction of the synthetic reactions}

of “Primase” and

“Polymerase-III” is opposite to the direction of

primosome movement

2. Elongation

_{Lagging strand or discontinuous DNA}

（

5

）

Elongation

DNA polymerase III.

Leading strand:

continuous replicate Lagging strand:

discontinuous

Primer removal by DNA Pol I 5’-3’ exonuclease

activity

• _{When the new okazakii fragment is complete, the RNA}

primer is removed by DNA polymerase-I and is replaced with DNA by the same enzyme. The remaining nick is sealed by “DNA ligase”.

Interaction of “Tus protein” with termination

sites stops DNA replication:

21-28

E.Coli DNA contain termination (TER) sites, which bind a specific protein called Tus. This protein may act to

stop replication by preventing “Helicase (DnaB) from unwinding duplex DNA, thereby interrupting the

function of the growing fork. Replication of the circular E.coli chromosome produces

Termination has a “Topological

problem”.

21-29

Termination results in catenation of the molecule

Decatenation is brought about by Type II topoisomerase, DNA