Plasmid replication -methods & types

bacterial plasmid and plasmid vector replication methods

1. PLASMID NEERAJA. A 15618

2. DEFINITION a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. They are autonomously replicating pieces of DNA

3. INTRODUCTION Used in the study of replication and DNA repair, due to its small size Dispensability to host Easy manipulation Responsible for the evolution of drug resistance & evolution of complex interaction with animal or plant hosts

4. REPLICATION Replication varies depending on The initiation of DNA replication & Leading and lagging strand synthesis are coupled or uncoupled

5. REPLICATION INITIATION Replication initiation depends on a sequence – plasmid origin of replication ( ori ). Basic replicon - the minimal sequence that supports replication, preserving the regulating circuitary . Minimal replicon – the minimal portion supporting the plasmid replication

6. CONTD.. Rep proteins – plasmid encoded initiators of replication. Some theta plasmids rely on host initiation factors for replication. Rep recognition sites consists of direct repeats or iterons. Rep binding of ori iterons leads to the formation of nucleoprotein complex that opens up DNA duplex at A+T rich segment.

7. CONTD... ITERONS- Iterons are directly repeated DNA sequences which play an important role in regulation of plasmid copy number in bacterial cells. It is one among the three negative regulatory elements found in plasmids which control its copy numbe r

8. DUPLEX MELTING&REPLISOME ASSEMBLY Two types- depending on replicon Dependent on transcription Mediated by trans-acting proteins

9. Rolling Circle Model Of DNA Replication

10. Replication in eukaryotes is bidirectional, this type is unidirectional. Ideal example of this type is the circular plasmid of bacteria, as it happens only in circular genomes. Initiation Initiates by the production of nick on one of the two strands producing free 3́-OH and 5́ phosphate ends, by the action of: Helicase Topoisomerases Single stranded binding proteins (SSBPs). Helicase Bacterial Plasmid SSBPs

11. ELONGATION For Elongation, the DNA polymerase III binds to the 3́-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling circle model . As the elongation proceeds, the 5́end will be displaced and will grow out like a waving thread.

12. DNA Polymerase III Broken Strand Origin Point

13. At the point of termination, the linear DNA molecule is cleaved from the circle, resulting in a double stranded circular DNA molecule and a single-stranded linear DNA molecule. The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule. Origin Point Click to add text TERMINATION

14. STRAND DISPLACEMENT

15. MECHANISM Parental duplex strand have two single stranded replication initiation sites ( ssi -A & ssi -B) Vertical lines show hybridization between DNA strands. Duplex melting is through RepC and RepA helicase, allowing two sites to form hair pin.

16. Contd.. The hair pin base are recognized by RepB', which initiates the synthesis of RNA primer. Extension of 3'-OH of primer by POL-III is assisted by RepA. Two D loops are formed as parent strands are displaced and dissociated from each other,leaving ssDNA intermediates (no H-bonding). Synthesis continues in both direction extending area of D loop formation.

17. Contd.. Elongation is completed and termination occurs at ssi sites in which replication began. At this point, the newly synthesized daughter strands are restored. Segregation: the two daughter strands are ligated, resulting in two DNA duplexes each containing a parental strand and a daughter strand

18. STRAND DISPLACEMENT