dna replication.pptx by Sahil khan sahab

Biochemistry

1. DNA REPLICATION By: rinku yadav

2. DNA replication  is the biological process of producing two identical replicas of DNA from one original  DNA  molecule. DNA replication occurs in all  living organisms  acting as the most essential part for  biological inheritance .

3. Basic requirement of replication : dATP dGTP dCTP dTTP   template: is defined as  a single-stranded nucleic acid – a DNA or RNA polymer made of nucleotides – that is used to synthesize a new DNA, RNA or protein polymer .

4. Primer: is  a short nucleic acid sequence that provides a starting point for DNA synthesis . primers are short strands of RNA. A primer must be synthesized by an enzyme called primase ,

5. helicase: unwind the double-stranded DNA molecule by breaking the hydrogen bonds between the complementary base pairs, allowing the DNA strands to separate . DNA C proteins then  help DNA helicase (DNA B) load onto the origin correctly . Histone-like proteins ( maintaining DNA architecture and regulating DNA transactions such as replication, recombination/repair and transcription .

6. STEPS OF REPLICATION separation of DNA strands: The regulation of DNA replication origins starts in the G1 phase of the cell cycle The Origin of replication in DNA stand the sequence present in A=T

7. helicase enzyme going to DNA strand and search the sequence of A=T (origin of replication) Break the hydrogen bound and formed (Y) shape structure called replication fork

8. Step -2 Synthesis of new strand DNA polymerase responsible for adding new nucleotides synthesis new DNA stand DNA Polymerases also possess exonuclease activity , that cuts incorrectly added nucleotides, and allows the DNA replication to happen without errors. DNA polymerase work as a single stand DNA polymerase the 2 nd condition gives work as a 3 prime to 5 prime

9. Single strand binding proteins (SSB) protein are DNA binding proteins, that binds to single-stranded DNA to help DNA replication. SSB proteins prevent the hardening of strands during DNA replication. It also protects strands from nuclease degradation and prevents the rewinding of DNA . 

10. DNA topoisomerase DNA topoisomerase is a class of enzymes that release helical tension during transcription and replication  Class Ι DNA topoisomerase makes a single-stranded break to relax the helix and progress the process. Class ΙΙ DNA topoisomerase break both the strands of DNA helix, this class of topoisomerases is also very important during the cell cycle for the condensation of chromosomes.

11. DNA PRIMASE DNA primase combined with DNA segment The DNA polymerase break the RNA primer and remove the RNA fragment or filling the DNA fragment

12. Ligase : These are fill the gape know it is called glowing enzyme with the help of phosphodiester bound (binding the Okazaki fragment) DNA ligase is used in both DNA repair and DNA replication

13. DNA polymerase check the stand like formation of stand formed properly This process called proof reading and DNA repair

14. Leading strand : The parent strand that runs in the 3' to 5' direction  can be easily read by DNA polymerase and as a result, this newly synthesized strand of DNA is called the leading strand. The leading strand is synthesized continuously in the forward direction (same direction as the movement of the replication fork).

15. lagging strand is  the strand of daughter DNA that is synthesized discontinuously in DNA replication .

16. Okazaki fragments are  short sections of DNA formed at the time of discontinuous synthesis of the lagging strand during replication of DNA . It is essential as it allows for the synthesis of both the daughter strands required for cell division.

17. SEMICONSERVATIVE REPLICATION THEORY According to the semiconservative model,  after one round of replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA.

18. Steps in DNA Replication Formation of Replication Fork   Initiation Elongation  Termination

19. DNA Replication in Prokaryotes the small size of the genome and the mutants that are available.  E. coli  has 4.6 million base pairs single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. approximately 1000 nucleotides are added per second.

20. In prokaryotes, three main types of polymerases are known: DNA pol DNA pol I : Exonuclease activity removes RNA primer and replaces with newly synthesized DNA DNA pol II : Repair function DNA pol III :Main enzyme that adds nucleotides in the 5'-3' direction

21. specific nucleotide sequences called origins of replication where replication begins. In  E. coli,  which has a single origin of replication. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called  helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. the DNA opens up, Y-shaped structures called  replication forks  . Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds.  

22. Single-strand binding the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5' to 3' RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand

23. DNA replication steps DNA unwinds at the origin of replication. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling. Primase synthesizes RNA primers complementary to the DNA strand.

24. 6.DNA polymerase starts adding nucleotides to the 3'-OH end of the primer. 7.Elongation of both the lagging and the leading strand continues. 8. RNA primers are removed by exonuclease activity. 9.Gaps are filled by DNA pol by adding dNTPs. 10.The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.

25. DNA Replication in Eukaryotes :D NA replication in eukaryotes occurs in three stages: initiation, elongation, and termination.

26. Eukaryotic DNA Replication- Features, Enzymes, Process, Significance DNA replication is the process by which an organism duplicates its  DNA  into another copy that is passed on to daughter cells. Replication occurs before a cell divides to ensure that both cells receive an exact copy of the parent’s genetic material.

27. Features of Eukaryotic DNA Replication Replication is bi-directional and originates at multiple origins of replication (Ori C) in eukaryotes. DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand. It occurs only in the S phase and at many chromosomal origins. Takes place in the cell nucleus. Synthesis occurs only in the 5′to 3′direction.

28. Individual strands of DNA are manufactured in different directions, producing a leading and a lagging strand. Lagging strands are created by the production of small DNA fragments called Okazaki fragments that are eventually joined together. Eukaryotic cells possess five types of polymerases involved in the replication process.

29. THE ENZYMES OF DNA REPLICATION Helicases:  Unwind the DNA helix at the start of replication. SSB proteins:  Bind to the single strands of unwound DNA to prevent reformation of the DNA helix during replication. DNA Polymerases : Eukaryotic cells contain five different DNA polymerases; α, β, γ, δ & ε. DNA polymerases α and δ replicate chromosomal DNA, DNA polymerases β and ε repair DNA, and DNA polymerase γ replicates mitochondrial DNA.

30. DNA polymerase α and δ synthesize the lagging strand, via Okazaki fragments. The RNA primers are synthesized by DNA polymerase α which carries a primase subunit. DNA polymerase δ synthesizes the leading strand. Telomerase , a DNA polymerase that contains an integral RNA that acts as its own primer, is used to replicate DNA at the ends of chromosomes (telomeres).

31. DNA topoisomerase I:  Relaxes the DNA helix during replication through creation of a nick in one of the DNA strands. DNA topoisomerase II:  Relieves the strain on the DNA helix during replication by forming supercoils in the helix through the creation of nicks in both strands of DNA. DNA ligase:  Forms a 3′-5′phosphodiester bond between adjacent fragments of DNA.

32. Process of Eukaryotic DNA Replication Replication of each linear DNA molecule in a chromosome starts at many origins, one every 30–300 kb of DNA depending on the species and tissue, and proceeds bi-directionally from each origin. At each origin, a replication bubble forms consisting of two replication forks moving in opposite directions . The DNA replicated under the control of a single origin is called a replicon. DNA synthesis proceeds until replication bubbles merge together.

33. At the origin, enzymes unwind the double helix making its components accessible for replication.  The helix is unwound by helicase to form a pair of replication forks. The unwound helix is stabilized by SSB proteins and  DNA topoisomerases . The RNA primers required are made by DNA polymerase α which carries a primase subunit. DNA polymerase α initiates synthesis of the lagging strand, making first the RNA primer and then extending it with a short region of DNA.

34. DNA polymerase δ then synthesizes the rest of the Okazaki fragment. The leading strand is synthesized by DNA polymerase δ. The leading strand is synthesized continuously in the 5′to 3′ direction while the lagging strand is synthesized discontinuously in the 5′to 3′ direction through the formation of Okazaki fragments . At the completion of synthesis, DNA ligase seals the breaks between the Okazaki fragments as well as around the primers to form continuous strands.

35. DNA Proofreading In eukaryotes only the polymerases that deal with the elongation (delta and epsilon) have proofreading ability (3’ → 5’ exonuclease activity). If an error is detected, the erroneous base is removed via 3′to 5′exonuclease activity replaced with the correct base. Excision repair : Removes pyrimidine dimers formed by UV rays or other mutated bases and replaces them.

36. Significance of Eukaryotic DNA Replication DNA replication is a fundamental genetic process that is essential for cell growth and division.  DNA replication involve the generation of a new molecule of nucleic acid, DNA, crucial for life. DNA replication is important for properly regulating the growth and division of cells.  It conserves the entire genome for the next generation.

37. DNA Repair Types and Mechanisms Nucleotide excision repair NER deals with bulky adducts and cross-linking lesions caused by UV radiation or chemical exposure.  NER removes a fragment of nucleotides containing the damaged lesion and synthesizes a new DNA strand using the undamaged strand as a template.

38. NER consists of two pathways: Global Genome NER (GG-NER) repairs bulky damages throughout the entire genome, including regions that are not actively transcribed.  Transcription-Coupled NER (TC-NER repairs damage that occurs on the transcribed DNA strand.  Mutations in NER pathway genes can lead to disorders such as xeroderma pigmentosum (XP) and certain other neurodegenerative conditions .

39. Inhibitors of Replication 1. Aphidicolin: Reversible inhibitor of eukaryotic nuclear DNA replication. Blocks the cell cycle at early S phase .Specific inhibitor of DNA polymerase in eukaryotic cells and in some viruses of animal origin. Apoptosis inhibitor/inducer. . Emetine dihydrochloride: Irreversibly blocks protein synthesis by inhibiting the movement of ribosome along the mRNA. Inhibits DNA replication in the early S phase as determined by cytometric analysis

40. 5-Fluorouracil A pyrimidine antimetabolite. Prevents the biosynthesis of thymidine during DNA synthesis. Inhibitors of Replication         5-Fluorouracil:  A pyrimidine antimetabolite. Prevents the biosynthesis of thymidine during DNA synthesis. Ganciclovir:  Nucleoside analog used to disrupt DNA replication.   Lomefloxacin  :A fluoroquinolone antibiotic that inhibits DNA gyrase. Mitomycin  - C Inhibitor of DNA synthesis, nuclear division and cancer cells. Penciclovir:  Nucleoside analog that blocks DNA replication.