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Replication of DNA

Replication of DNA. DNA replication. Synthesis of DNA is semidiscontinuous and bidirectional Replication fork is formed to start the replication Different substances and enzymes are required for the replication RNA primer Primase DNA polymerase I, II & III DNA helicases Topoisomerases

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Replication of DNA

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  1. Replication of DNA

  2. DNA replication • Synthesis of DNA is semidiscontinuous and bidirectional • Replication fork is formed to start the replication • Different substances and enzymes are required for the replication • RNA primer • Primase • DNA polymerase I, II & III • DNA helicases • Topoisomerases • Ligases • Single stranded binding proteins (SSB)

  3. Alternative models of DNA replication

  4. The Chemistry of DNA Synthesis • The Mechanism of DNA Polymerase • The Replication Fork • The Specialization of DNA Polymerases • DNA Synthesis at the Replication Fork • Initiation of DNA Replication • Binding and Unwinding • Finishing Replication

  5. Initiation of replication, major elements: • Segments of single-stranded DNA are called template strands. • Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. • Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). • DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis) • Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides. • Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer. • The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase. • Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process.

  6. DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand: • Unwinding of any single DNA replication fork proceeds in one direction. • The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’. • Solution: DNA is made in opposite directions on each template. • Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement. • continuous • requires a single RNA primer • Lagging strand synthesized 5’ to 3’ in the opposite direction. • semidiscontinuous (i.e., not continuous) • requires many RNA primers

  7. The replication fork • The junction between the newly separated template strands and the unreplicated duplex DNAis known as the replication fork

  8. Origin of replication (e.g., the prokaryote example): • Begins with double-helix denaturing into single-strands thus exposing the bases. • Exposes a replication bubble from which replication proceeds in both directions.

  9. Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins: 5’ SSB Proteins Okazaki Fragments ATP 1 Polymerase III 2 Helicase + Initiator Proteins 3 Lagging strand 3’ primase base pairs 5’ Polymerase III RNA primer replaced by polymerase I & gap is sealed by ligase 5’  3’ Leading strand RNA Primer 3’

  10. Both strands of DNA are synthesized together at the replication fork. Leading strand Okazaki fragments Replication fork Lagging strand

  11. DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond (Fig. 3.7)

  12. Model of replication in E. coli (Fig. 3.5)

  13. Model for the events occurring around a single replication fork of the E. coli chromosome

  14. Model for the events occurring around a single replication fork of the E. coli chromosome

  15. Replication of circular DNA in E. coli: Two replication forks result in a theta-like () structure. As strands separate, positive supercoils form elsewhere in the molecule. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

  16. DNA replication in eukaryotes: Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system Cells must be large enough, and the environment favorable. Cell will not enter the mitotic phase unless all the DNA has replicated. Chromosomes also must be attached to the mitotic spindle for mitosis to complete. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).

  17. Eukaryotic enzymes: • Five DNA polymerases from mammals. • Polymerase  (alpha): nuclear DNA replication, no proofreading • Polymerase  (beta): nuclear, DNA repair, no proofreading • Polymerase  (gamma): mitochondrial DNA replication, proofreading • Polymerase  (delta): nuclear, DNA replication, proofreading • Polymerase  (epsilon): nuclear, DNA repair, proofreading • Different polymerases for nucleus and mtDNA • Some proofread; others do not. • Some used for replication; others for repair.

  18. Each eukaryotic chromosome is one linear DNA double helix • Average ~108 base pairs long • With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. • Solution ---> DNA replication initiates at many different sites simultaneously.

  19. The initiation of a new strand of DNA require an RNA primer • Primase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence. • DNA Pol can extend both RNA and DNA primers annealed to DNA template

  20. RNA primers must be removed to complete DNA replication A joint efforts of RNase H, DNA polymerase & DNA ligase

  21. Topoisomerase removes supercoils produced by DNA unwinding at the replication fork

  22. DNA proofreading • After the synthesis of DNA strand DNA polymerase enzymes proofreads for any mistakes present in newly synthesized DNA.

  23. Types of DNA damage • Single base alteration: e.g. deamination of single base • Two base alteration: e.g. UV light induced formation of thymine dimer • Chain breaks: e.g. due to ionization • Cross links: formed between bases of same or opposite strands

  24. Repair of DNA damage • Thymine dimer formation block the DNA replication • UV specific endonuclease can recognize the dimer and DNA polymerase removes that one • Xeroderma pigmentosa is rare autosomal recessive genetic disease due to the defect in the repair of DNA damage

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