Replication Origins:

 

Basically, replication initiates, whatever may be type of DNA it is, at a specific site or locus called ORIGIN (or Ori) and terminates the replication at a specific site, called Termination site, also called TER sites. The structure, location and the number of Origins and Ter site vary from one system to the other.  Origins can be recognized by the site of replicating bubble and fork movement for direction of replication.  Every genomic DNA contains an origin site.  Lower organisms like E.coli have only one Ori, but higher eukaryotic systems have multiple ORIs’.  The size of replicon and the structure and location of Origin vary from one genome to the other.  This site is involved in the binding of specific proteins, which results in the unwinding of the DNA into open structure called replication eye or Replication bubble, on to which a variety of proteins bind and initiate replication.   Depending upon the kind of origin, the replication from the origin can be bi-directional or unidirectional with some variations in each of the two.

 

Types:  Mutants can delineate the structure and function of ORIs.  Understanding of ORIs is very important for amplification of recombinant DNA under in vitro conditions, whether it is for gene cloning or other genetic manipulations.

 

A DNA containing an Ori site and a Ter site is called a Replicon, the unit of replication, irrespective of the length of the genomic DNA.  E.coli DNA consists of one Replicon.  In eukaryotes as the size of DNA is thousand times the size of E.coli DNA; it is organized into several nucleo proteinaceous threads called chromosomes.   Sheer size of the DNA in eukaryotes (EKs) is so high; each chromosomal DNA contains several replication origins and termination sites, however the Ter sites are not very well defined. Eukaryotic Replicons, have devised replication mechanism with multiple ORIs and TER sites; so as to achieve the completion of replication within a time period.

 

 

 

 

Replicons, a Comparative Account:

 

Species

Genome size (bp)

Replicon numbers

Size of a Replicon

Rate of replication (bp)/min

E.coli

4.6 x 10^6

1

4.6 x ^6

50000 to 60000

Yeast (n=16)

1.4 x 10^7

500

40,000

36,000

Fruit fly (n=4)

1.4 x 10^8

3500

40,000

2800

House fly (n=4)

8.6 x 10^8

4000

45,000

2800

X.laevis (n=)

3.1 x 10^9

15000

200kbp

2500

 

 

 

 

 

Mus musculus

2.6 x 10^9

25000

150kbp

2200

Homo sapiens

3.0 x 10^9

25-30 000

200kbp

2800-3000

 

 

A list of Origins:

 

ORI-Name

Species

Replication direction

Size of the Origin

Ori-C

E.coli

Coupled Bidirectional

250 bp

Ori-Col.E 1

Col.E1 plasmid

Unidirectional, leading strand first initiated

 

Ori Ti

Ti plasmid from Agrobacterium tumefaciens

Bidirectional

 

Ori PSc 100

PSc plasmid

 

 

Ori Pi

Pi phage

Bidirectional

 

Ori Col V

Plasmid Col.V-k30

 

 

Ori-R 300

Drug resistant plasmids

 

 

Ori-alpha

Plasmid alpha, Ori-alpha      

Unidirectional

 

 

Ori-y  

Ori-y  

Sequentially bidirectional, one at ori and second at n’pas

 

Ori-F  

Ori-F   from F plasmids

Bidirectional

 

 

Ori-R1

R1 and R100

Unidirectional, lagging strand first initiated

 

Ori-S 

HSV virus

Sequentially bidirectional, First initiation at ori, then second at n’pas

 

Ori-P

EBV

 

 

Epstein Barr Viral

Ori-BPV

BPV

 

 

Ovine Papilloma Viral

Ori Rk2

RK2 plasmid

 

 

Ori-ADV

Adeno virus, at the end of linear DNA

Unidirectional

 

Ori-Rhi

Rhizobium plasmid

 

 

Ori-H

Mitochondria, H stands for Heavy strand replication

Unidirectional

 

Ori-L

Mitochondria, for replication of light strand

Unidirectional

 

Ori T7

Phage T7

Bidirectional/

Unidirectional

 

Ori lambda

Phage lambda

Coupled Bidirectional

 

Ori phi X

Origin for PhiX174 phage DNA

 

Unidirectional

Ori (+) located at 4299-4328= ~30 ntds.

Ori (-) located at 2220-2300 =~80ntds

Ori M13

Phage M13 (filamentous)

Unidirectional

Ori (+) located at5750-5850 (contain D and E loops)

Ori (-) located at 5600-5750(contain B and C loops)

 

 

 

 

 

 

 

Ori sites that Use Both Dna-A and Rep Proteins:

 

Plasmid DNA

Dna-A box

A/T (13mers)

Rep site (18-19 bp)

A/Y rich (5-11 bp)

GATC methyl sites

IHF (histone like factors)

PSc 101

1

2

5 x18 bp

None

1

Yes

Pi Ori-R

2

-

5 x 19 bp

5 x 7 bp

5

No

F, Ori-S

2

1

4 x 19 bp

 4 x 8 bp

1

-

Ori-col-V-K30

2

-

5 x 19 bp

5 x 11 bp

-

-

E.Coli

4

3

-

3 x 19 bp

1

Yes

Ori-gamma (R6K)

1

1

7 x22 bp

3 x 10 bp

1

Yes

 

           

 Kinds and Components of Origins:

 

Comparative structural components of different Origins:

 

E.coli-13----13----13R1-IHF-M-R2-Fis-R3-R4,R=A=9mer [13mer=GATCTNTTNTTT, 9mer=TGTGAATA],

 

B.subtilis-----16-----16---16-----A-9----A-9----A-9----A-9 [13mer=GATACCTTANTTTTC],

 

Pseudomonas---12-----12---12------A-9----A-9----A-9----A-9 [13mer=GAAGAGGTT/AAT/A—T/A],

 

Lambda     [11 AT rich] x 4---------- [19] x 4; TTNTCTTTTGT,

O-lambda binds to 19 mer regions and lambda P-proteins bind AT rich 11bp region

 

O82              [11 AT rich] x 2-------- [19] x 4; TTGTCTTTTGT, O-82 bind

O80             [6 AT rich] x 3----------- [19] x 4; TCTTGT,

 

 

E.coli origin loci:

 

Replication origin in E.coli DNA is located at 84.3mpu in between genes like gid A and mio C.

 

--uncB—UncI—gidB-gidA---<OriC>-mioC-asnC<>asnA---dATA                 

 

E.coli: -0--13----13----13—<R1-IHF-<M-R2>-<Fis>-<R3-R4>--340bp.

 

R=A,  [3x13mer=GATCTNTTNTTT],  [4x9mer=TGTGAATA],

 

 

Black shaded rectangles are GATC sequences; R1,R2,R3 and R4 are the DnaA binding sites; IHF-alpha and beta, integration host factor are histone like protein, IciA inhibitors (Inhibitor of chromosome initiation), ROB Right arm binding sites; R3 is weak binding site.

 

http://oregonstate.edu/

 

 

 

 

The above figure shows E.coli Ori C components, the binding of DNA to 9-mer sequences lead to melting of the DNA into replication bubble.

 

E.coli- Pre-primosomes

 

http://oregonstate.edu/

 

           

 

The diagram shows origin components of different DNAs from different plasmids and viruses; http://embor.embopress.org/

 

 

 

Properties of some Plasmid DNA and their Origins:

 

Plasmid

Ori

Size of the plasmid (Kbp)

Copy no.s/cell

Required proteins

Host range

PSC 101

 

9

4-6

Rep-A (38kd)

Limited

F

Ori-S

90

1-2

Rep-E (29kd)

Limited

Col V K30

Ori V

90

1-20

 

Limited

R 300 B

 

9

10-20

 

Limited

R1, R100

 

90

1-2

Rep-A (33kd)

Limited

R6K

Ori-R

38

1-11

36KD

Limited

RK2

 

56

5-8

Trf-A (32-43kd)

Broad host range

Col-E1

Col E1 ori

6kbp

10-20 (100)

 

 

Ti

Ori-T

150-500

1-2

 

 

Rhi

Ori-rhi

40-50

1-2

 

 

 

Replication Termination Regions in E.coli:

 

When replication progresses; it has to end at some region.  In the case of E.coli replication progresses in bidirectional and the replication forks meet at a common point called termination site or TER region.  This is about 36 kbp and consists of six 23bp long consensus sequences; they are located almost opposite to origin region and oriented in opposite orientations, the are Left- < E <D <A : : :  >C > B > F, the sequences- AATTAGTATGTTGTAACTAAAGT.  This region is recognized by TER utilizing substance called TUS protein of 36Kd and they bind and prevent the movement of Helicase into double stranded daughter DNA molecule, thus prevent the second round replication.

 

The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (Ori C), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from .

The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (Ori C), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from; The diagram shows both Origin sites and TER sites; http://proteopedia.org/