Origin of replication, replication fork, enzymes

MICROBIOLOGY

1. PRESENTED BY: Mousami Jaria St. George College of Management and Science MSc Microbiology Semester 2 ORIGIN OF REPLICATION , REPLICATION FORK, ENZYMES INVOLVED IN REPLICATION

2. DNA replication is also known as semi conservative replication. It is the process by which DNA is essentially doubled i.e makes its exact copies. It is an important process that takes place within the dividing cell . DNA is copied during the S phase of interphase . Replication is the process of formation of exact carbon copies of a substance. DNA REPLICATION

3. DNA STRUCTURE

4. DNA replication can occur by three possible methods: Conservative replication : The parent structure remains intact. The replica is a completely new structure. Disruptive replication : The parent structure fragments and two new structures are formed afresh. Semiconservative replication : One half of the parent structure passes into each replica and the second half is built anew. DNA structure proposed by Watson and Crick (1953) was based on its semiconsevative replication.

5. DNA replication is a complex multistep process that require a number of enzymes , protein factors and ions. ORIGIN OF REPLICATION : Replication begins at a particular region of DNA, characterized by primarily three types of structures: Sites for binding of proteins mainly intiation and auxillary proteins. A characteristically AT rich region that is unwound Sites & structural properties involved in regulating initiation. MECHANISM OF DNA REPLICATION

6. It is called origin of replication or ori . Prokaryotes have single origin of replication while eukaryotes have several of them. DNA of prokaryotes functions as a single replicating unit called ‘’ replicon ’’. DNA of eukaryotes possess a number of replicons or replicating units. The use of vector for recombiant DNA procedure is meant for providing origin of replication. Replication proceeds on both sides from Ori , this is called bidirectional replication . Unidirectional replication is rare.

7. ACTIVATION OF DEOXYRIBONUCLEOTIDES : 4 types of deoxyribonucleotides – dAMP , dGMP , dTMP , dCMP . With the help of energy , phosphate and enz . phophorylase the nucleotides are changed in triphosphate state i.e dATP , dGTP , dTTP , dCTP .

8. INITIATION: Origin of replication is recoganized by recognition complex. It attracts enzymes . Enzyme helicase unwinds the DNA helix and unzips the two strands of DNA by breaking hydrogen bonds. The separated strands become stabilized in this condition with the help of Single strand binding proteins or SSBPs. Unwinding creates tension which is released by cutting and resealing enzymes topoisomerases I &II. FORMATION OF REPLICATION FORK

9. Topoisomerase II of prokaryotes is also called gyrase . It functions both as helicase and topoisomerase . Unzipping creates a Y-shaped configuration called replication fork , forms within the nucleus during DNA replication. It is created by helicases , which break the hydrogen bond holding the two DNA strands together The resulting structure has two branching “ prongs ”, each made up of single strand DNA . These two strands serve as the template for leading and lagging strand , which will be created as DNA polymerase matches complementary nucleotides to the templates, the templates may be properly referred to as the leading strand template and the lagging

10. strand template. DNA is always synthesized in 5’ to 3’ direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork , a major issue is how to achieve synthesis of new lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork .

11. The leading strand is the strand of new DNA which is being synthesized in the same direction as the growing replication fork. The polymerase reads the leading strand template and adds complementary nucleotides to the new leading strand on continuous basis . LEADING STRAND

12. The lagging strand is the strand of new DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation , replication of the lagging strand is more complicated as compared to leading strand. As a consequence, DNA polymerase on this strand is seen to lag behind the other strand. LAGGING STRAND

13. The lagging strand is synthesized in short , separated segments. On the lagging strand template , a primase reads the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming okazaki fragments The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase .

14. Okazaki fragments are small stretches of DNA with 1000-2000 base pairs in length. Named after scientist Rejis Okazaki who discovered them in 1968. These fragments are synthesized by DNA polymerase. Okazaki fragments are joined together by enzyme DNA ligase OKAZAKI FRAGMENTS

15. A large number of enzymes are required for DNA replication The main enzyme which takes part in combining deoxyribose nucleotides to form new DNA strands is called DNA dependent DNA polymerase The other enzymes required for DNA replication are- primase , topoisomerase , helicase , single strand binding proteins,DNA ligase etc. ENZYMES INVOLVED

16. These enzymes copy DNA sequences by using one strand as a template. The reaction catalysed by DNA polymerases s the addition of deoxyribonucleotides to a DNA chain . Prokaryotes have three types of DNA polymerases- I , II, III . DNA polymerase I ( also called Kornberg enzyme ) is used in proof reading, whereas, DNA polymerase II is a specialized repair enzyme. DNA polymerase III actually takes part in replication. Eukaryotes have 5 types of DNA polymerases. DNA POLYMERASES

17. It is called DNA unwinding protein. Binds to single stranded regions of DNA to prevent premature annealing, to protect ss DNA from being digested by nucleases. To remove secondary structure from DNA to allow other enzymes to function effectively upon it. Major function is to prevent recoiling of DNA strands after its unwinding by helicases . SINGLE STAND BINDING PROTEINS (SSBPs )

18. It is strand of nucleic acid that serves as a starting point for DNA synthesis. DNA polymerases adds new nucleotides to existing strand of DNA. Replication starts at the 3’ end of the primer, and copies the opposite strand. PRIMASE: It is a type of RNA polymerase which creates RNA primer. RNA primer functions as 5’ end of the new strand( to be synthesised ). PRIMER

19. Primase is of key importance because no known DNA polymerases can initiate the synthesis of DNA strand without an initial RNA or DNA primer. DNA GYRASE Simply referred as gyrase . It relieves strain while ds DNA is being unwound by helicase . This cause negative supercoiling of DNA. The ability of gyrase ( also topoisomerase ) to relax positive supercoiles allows superhelical tension ahead of the polymerase to be released so that replication can continue.

20. UNTANGLING DNA WITH GYRASE

21. They are often used to separate strands of DNA double helix. They are proteins which are involved in unwinding of DNA molecule, to provide a ssDNA for replication , transcription and recombination. TOPOISOMERASE They are a group of enzymes which control supercoiling of DNA thereby maintaining a superhelical tension . They are of two types – type I and type II HELICASE

22. Responsible for connecting DNA segments during replication, repair and recombination They catalyze the formation of alpha- phosphodiester bond between two DNA chains This enzyme requires the free OH group at 3’ end of the other DNA strand and phosphate group at 5’ end of the other. DNA LIGASE

23. LIGATION OF TWO STICKY END DNA FRAGMENT

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