Rolling circle mechanism ppt

rolling circle present in plasmid

1. Mechanism of Rolling C ircle Mrs. Praveen Garg Asst. Prof., VITS College, Satna , M.P .

2. Introduction Rolling circle replication is a process of unidirectional nucleic acid replication. It can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmid. Eukaryotic also replicate their DNA/RNA via this methods. Widely used in molecular biology & biochemical nanotechnology, especially in the field of biosensing (as a method of signal amplification).

3. DNA rolling circle replication has five Steps Circular ds DNA will be nicked. 3’ end is elongated using unnicked DNA as leading strand (template) 5’ end displaced is as a lagging strand and is made double stranded DNA via a series of OKAZAKI fragments. Replication of both unnicked and displaced ss DNA. Displaced DNA circulates and synthesis its own complementary strand.

4. Thrre steps of Rolling Circle Replication (1) Initiation In the process of initiation, three enzymes works together such as: Helicase Topoisomerase Single stranded binding proteins (SSBPs) These enzymes bind with phosphate ends of nick strand.

5. (2) Elongation In the process of elongation, 3’OH group of broken strand, work as a template. The polymerase enzyme help to move in a circle for elongation, due to which it is named as rolling circle mechanism. 5’ end will be displaced and grow like as a thread.

6. (3)Termination 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 DNA molecule. The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.

7. Rolling circle DNA replication Rolling circle replication is initiated by protein coded by the plasmid or bacterial DNA, called initiator protein. This protein nicks one strand of the double stranded DNA at a site called double stranded origin or DSO. This protein bind to 5’ phosphate end of the nicked strand and free 3’ OH end is serve as a primer for DNA synthesis by DNA polymerase enzyme. 5’ end displaced and grow like as a thread with the help of helicase and single stranded binding proteins. Continued DNA synthesis can produce multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series called a  concatemer . These linear copies can be converted to double-stranded circular molecules through the following process: First, the initiator protein makes another nick in the DNA to terminate synthesis of the first (leading) strand.  

8. Replication starts at ORI region by a initiator Rep A Region bind with DNA polymerase and Replication starts

9. Helicase also attach to the region Progeny positive strand synthesis

10. Positive strand also synthesis its complementary strand

11. Breakage is joining by ligase Progeny strand can also synthesize another strand of DNA

12. Various steps involve in this process: Firstly, Initiator protein Rep A bind to nicked 5’ OH end of ssDNA at ori site . After that DNA Polymerase enzyme bind with 3’P end of ds DNA or ori site. Then helicase enzyme attach with RepA protein and start replication process. In this process 5’ end of ssDNA elongated with the help of single stranded binding protein. After completing new ssDNA synthesis, new strand separate with the single stranded binding protein from the nicked region and nick place sealed by DNA ligase enzyme. At last single stranded DNA also synthesize its complementory strands.

13. Example: Viral DNA : Some DNA viruses replicate their genomic information in host cells via rolling circle replication .  Human herpes virus Human papilloma virus Gemini virus Viral RNA: Some RNA viruses and viroids also replicate their genome through rolling circle RNA replication. Pospiviridiae family   Avsunviridiae family

14. Y shaped rolling circle mechanism

15. Ө Shaped rolling circle mechanism

16. Thank you