Plasmid DNA Replication:

 

Col. E plasmid Replication

 

Plasmids are extra chromosomal DNAs, exist as ds circular molecules.  The size and structural features and replication modes vary from one species of plasmid to the other.  Some plasmids produce their own components for replication and others heavily dependent on host factors and enzymes.  Many such episomal DNAs have been identified by their specific features such as, for their drug resistant features called R-plasmids, self-transmissibility F-plasmids, and antibiotic resistant plasmids and anti bacterial plasmids like colicin plasmids.  Some plasmids resist to heavy metals, UV radiations, some produce restriction enzymes and some produce products that are involved in cellular metabolism. Agrobacterium plasmid called Ti plasmid (= 400kbp) is unique for its ability to transfer a segment of its own DNA called T-DNA into host plant cells, without the bacteria getting into the cell and induce tumors; it is an unique plasmid.

 

Characteristic Features of Some Plasmids:

                       

Plasmid

Size (kbp)

Self transmissibility

Copy nos.

Features

Col E1

6

No

10-20

Colicin^+

R300 B

9

No

10-20

 

Psc-101

9

No

4-6

TcR

F^+

90

Yes

1-2

F-pilius

Col V

90

Yes

1-2

 

Ent-p30

90

Yes

1-2

 

R1, R100

90

Yes

1-2

Drug resistance

R6K

38

Yes

10-20

 

RK2

56

Yes

5-8

Broad host range

Ti

500

Yes

1-2

Tumor inducing, transfers a part of its DNA into host cell

Rhi

40-50

Yes

1-2

Induces root nodules

 

 

 

Host coded proteins in Replication:

 

 

Hosts

RNAP

DNA pol-I

Dna-A

Dna-B

Dna-C

Dna-G

Dna-E

Dna-T

Col E1

+

+

+

+

+

+

+

 

PSC101

-

-

+

+

+

+

-

 

R1, R100

-

-

+

+

+

+

-

 

R6K

+

-

+

+

+

+

 

 

F^+

+

-

+

+

+

+

 

 

RK2

-

-

+

+

+

+

 

 

RSF 1010

-

-

-

-

-

+

 

 

 

 

 

 

 

 

 

 

 

           

 

Plasmid encoded proteins for initiation:

 

Plasmid

Gene

Protein

Mol.wt (KD)/ subunits

Function

PSC 101

Rep-A

Rep-A

38/ 1

Helicase, 3’>5’

R1

Rep-A

Rep-A

33/ 1

Helicase, 3’>5’

R100

Rep-A

Rep-A

33/ 1

Helicase 3’>5’

R6K

Pi-R

Pi-R

36/ 1

 

F^+

Rep-E

Rep-E

29

 

RK2

Trf-A

Trf-A

32-43

 

RSF 1010?

Rep-C

Rep-C

31

 

PT 181

Rep-C

Rep-C

38

 

 

 

 

Replication in Col E1.Plasmid DNA:

 

Initiation on leading strand:

 

 

Cole E1 –Colicin protein; http://www.chembio.uoguelph.ca/

 

 

 

 

 

 

 

 

 

 

 

ftp://ftp.tugraz.at/pub/Molekulare_Biotechnologie

 

 

 

 

Initiation on lagging strand:

 

·         In the replication bubble, just about 15 bp downstream of the ori site, on the lagging strand, small secondary structure forms (stem loop structure), which acts as the recognition site for the assembly of another primosome complex. 

 

·         Primosome assembly is hierarchical and starts with Pri- A.  

 

·         Pri-A, Pri-B and Pri-C first assemble on the n-pas site, soon Dna-B assisted by Dna-C joins the Pri-ABC group in ATP dependent manner.  At this point Dna-G, the primase joins. 

 

·         This primase is a monomer and resistant to Rifamycin unlike normal RNA-pol complex. The primase lays 11 ntds long RNA primer, whose ends are used by the DNA pol III Holozyme. 

 

·         Wherever the opened DNAs’ single strand is available, it is covered with SsBs.

 

·         Initiation, though takes place on both leading and lagging strands in the replication fork, the fork movement is only in one direction, and so replication is unidirectional.

 

Regulation of Initiation:

 

 

 

 

 

Regulation of plasmid colE1 copy numbers by antisense mechanism; http://www.sci.sdsu.edu/

 

Regulation of plasmid R1 copy numbers by antisense RNA: http://www.sci.sdsu.edu/

Inhibitory control over the RNA-II can be exercised only when the length of RNA II is just 15-200ntd long and before it hybridizes with the leading strand. 

 

 

 

 

Required Components Col.E1 Plasmid Replication:

 

Protein

Gene

Mol.wt (KD)

Subunits

Functions

 

Dna-A

Dna-A

50

1

Binds to box A

Pri-A

 

76

1

Primosome initiation

Pri-B

 

11.5

2, dimer

Primosome formation

Pri-C

 

23

1

Primosome formation

Dna-B

Dna-B

50

6, hexamer

Helicase, 5’ŕ3’ direction

Dna-C

Dna-C

29

1 to with Dna-B

Helps Dna-B in loading on to lagging strand in the fork region

SsBs

SsBs

19

4, tetramer

Bind to p-s-p backbone

RoM

 

7.5

Monomer

Facilitates RNA-I and RNA-II hybridization

RNA-pol

 

Pentamers

Produces RNA primers on leading strand

DNA-pol-I

 

103

1

Removes primers and fills the gap

DNA pol-III

Many

>512

>12

New Dna synthesis on both leading and lagging strands

DNA-ligase

 

 

1

Ligates ends