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DNA replication and repair

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“This structure has novel features which are of considerable biological interest.”

This may be the science most famous statement, which appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix.

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

Veröffentlicht in: Gesundheit & Medizin
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DNA replication and repair

  1. 1. DNA REPLICATION AND REPAIR Nirajan Shrestha Biomedical Research Institute Chonbuk National University Medical School
  2. 2. DNA: Introduction • DNA (Deoxyribonucleic acid) is the hereditary material in humans and almost all other organisms. • Most DNA is located in the nucleus (Nuclear DNA), but a small amount of DNA can also be found in the mitochondria. • The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases.
  3. 3. Structure of DNA • DNA exists as a double stranded molecule, in which the strands wind around each other, forming a double helix. • Double helix structure of DNA was proposed by James Watson and Francis Crick in April 1953, on the basis of X-ray diffraction model proposed by Rosalind Franklin and Maurice Wilkins. • Nine years later, in 1962, Watson and Crick shared the Nobel Prize in Physiology and Medicine with Maurice Wilkins.
  4. 4. • “This structure has novel features which are of considerable biological interest.” • This may be the science most famous statement, which appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix. • “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
  5. 5. Do not forget her….
  6. 6. • DNA is poly-deoxyribonucleotide that contain many mono-deoxy ribonucleotide covalently linked by 3’-5’ phosphodiester bond. • The chains are paired in anti-parallel manner. • In the DNA helix, the hydrophilic deoxyribose-phosphate backbone is on outside of the molecule, where as the hydrophobic bases are stacked inside. • Base pairing: A–T /G-C (H Bond)
  7. 7. DNA REPLICATION • DNA replication is the process of synthesis of two daughter DNA from single parental DNA molecule. • When the two strands of DNA double helix separated, each strand can contribute as a template for the daughter DNA. • In a single daughter DNA, one strand comes from parent and next is newly synthesized. • Hence, DNA replication is semi-conservative in nature.
  8. 8. Semi-conservative nature of DNA Replication
  9. 9. DNA Replication in Prokaryotes • The replication process described in this section were first known from studies of the bacterium E. coli. DNA synthesis in higher organisms is less well understood, but involves the same types of mechanisms with few exception. A. Separation of two complementary Strands B. Formation of replication fork C. Direction of DNA replication D. Synthesis of RNA primer E. Chain elongation F. Excision of RNA primer and their replacement by DNA G. DNA ligase action H. Termination
  10. 10. A. Separation of two complementary Strands • In order to replicate the parent DNA, they must first separate. • Replication begins at the point called “Origin of Replication”. • At the origin of replication, DnaA protein bind to specific nucleotide sequence. This energy requiring process cause the dsDNA to separate. As the dsDNA is unwound, a replication bubble forms.
  11. 11. B. Formation of Replication Fork • As the two strands unwind and separate, they form a “Y shaped” where active synthesis occurs. This region is called the replication fork. • DNA helicase unwinds the double helix. • The replication fork moves at the rate of 1000 nucleotides per second. • SSB protein helps to keep the strand separated. • As the two strands of the double helix are separated, a problem is encountered, namely, super-coiling in the region of DNA ahead of the replication fork.
  12. 12. • The accumulating positive supercoils interfere with further unwinding of the double helix • To solve the problem of super-coiling, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix. • These enzymes reversibly cut one strand of the double helix. They have both nuclease (strand-cutting) and ligase (strand- resealing) activities.
  13. 13. C. Direction of Replication • The DNA polymerases responsible for replication are only able to “read” the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new DNA strands only in the 5′→3′ (anti- parallel) direction. 1. Leading Strand: This strand is extended towards the replication fork and synthesized continuously. 2. Lagging strand: This strand is extended away from the replication fork and synthesized discontinuously in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. Okazaki fragments are named after the scientist who first discovered them.
  14. 14. Picture showing Replication Fork
  15. 15. D. RNA Primer • DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer, with a free hydroxyl group on the 3′-end of the RNA strand. • A specific RNA polymerase, called Primase (DnaG), synthesizes the short stretches of RNA (approximately ten nucleotides long) that are complementary and anti-parallel to the DNA template. • These short RNA Primer are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand.
  16. 16. E. Chain Elongation • DNA polymerases elongate a new DNA strand by adding deoxy- ribonucleotides, one at a time, to the 3′-end of the growing chain. • DNA chain elongation is catalyzed by DNA polymerase III. • The new strand grows in the 5′→3′ direction, anti-parallel to the parental strand . • Pyrophosphate (PPi) is released when each new deoxynucleoside monophosphate is added to the growing chain.
  17. 17. F. Excision of RNA primers and their replacement by DNA • DNA POL I removes the RNA primer and fills the gap between Okazaki fragments.
  18. 18. G. DNA Ligase Action • The final phosphodiester linkage between the 5′-phosphate group and the 3′-hydroxyl group on the chain is catalyzed by DNA ligase. • DNA ligase is an enzyme that catalyzes the sealing of nicks remaining in the DNA. • The joining of these two stretches of DNA requires energy, which in most organisms is provided by the cleavage of ATP to AMP + PPi.
  19. 19. H. Termination • Termination of DNA replication in E. coli is mediated by binding of the protein, TUS (Terminus Utilization Substance) to replication termination sites (Ter sites) on the DNA, stopping the movement of DNA polymerase.
  20. 20. Proof-reading Function of DNA POL III • The addition of an incorrect base can take place by a process called tautomerization. • If the wrong base is inserted then the bond is unstable. • DNA polymerase (I and III) have the ability to proofread, using 3' → 5' exonuclease activity. • When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.
  21. 21. Proofreading……………. • For example, if the template base is Thymine and the enzyme mistakenly inserts an cytosine instead of a Adenine into the new chain, the 3′→5′ exonuclease activity hydrolytically removes the misplaced nucleotide. The 5′→3′ polymerase activity then replaces it with the correct nucleotide. • The proofreading exonuclease activity requires movement in the 3′→5′ direction, not 5′→3′ like the polymerase activity. This is because the excision must be done in the reverse direction from that of synthesis.
  22. 22. Proofreading in figure
  23. 23. Eukaryotic DNA Replication • The process of eukaryotic DNA replication closely follow that of Prokaryotic DNA Synthesis. Prokaryotic DNA Replication Eukaryotic DNA Replication Single Origin of Replication Multiple Origin of replication Three types of DNA Polymerase Five types of DNA POL DNA POL I,II, III DNA POL α, β, Υ, δ and ε DNA POL III carries out both initiation and elongation Initiation is carried out by DNA polymerase α while elongation by DNA polymerase δ and ε DNA repair and gap filling are done by DNA polymerase I DNA polymerase β and ε performs this function RNA primer is removed by DNA polymerase I Removed by DNA polymerase β DNA POL Υ replicates mitochondrial DNA.
  24. 24. DNA Repair • Any manufacturing company tests its product in several ways to see whether its has been assembled correctly. Production mistakes are rectified before the item goes on market. The same is true for DNA synthesis. • DNA replication is incredibly accurate- only about 1 in 100,000 bases is added incorrectly. In addition to the proof- reading capabilities of the DNA polymerase, repair enzymes further assure the accuracy of DNA replication. This mechanism is called DNA repair. • A failure to repair DNA produces a mutation. Luckily, Cells are interestingly efficient at repairing the damage done to their DNA.
  25. 25. Agents that Damage DNA
  26. 26. Types DNA Repair DNA repair can be grouped into two major functional categories: A. Direct Damage reversal B. Excision of DNA damage
  27. 27. A. Direct Damage Reversal • It is the simplest repair mechanism. • Process in a single-reaction step • It involves enzymatic properties which binds to the damage and restores the DNA to its normal state. i) DNA photolyases ii) DNA- alkyltransferases
  28. 28. B. Excision of DNA damage I ) Base excision repair (BER) II) Nucleotide excision repair (NER) III) Mismatch repair (MMR)
  29. 29. I ) Base Excision Repair (BER) Base excision-repair of DNA • The enzyme uracil DNA glycosylase removes the uracil created by spontaneous deamination of cytosine in the DNA. • An endonuclease cuts the backbone near the defect • An endonuclease removes a few bases • The defect is filled in by the action of a DNA polymerase. • Finally, the strand is rejoined by a ligase.
  30. 30. • In Escherichia coli, there are three specific proteins, called UvrA, B and C, involved in lesion recognition. • This fragment is released by UvrD helicase action, generating a gap that is finally submitted to repair synthesis. II) Nucleotide excision repair (NER)
  31. 31. III) Mismatch Repair (MMR) • When a mismatch occurs, the proteins responsible for removal of mispaired nucleotides must be able to discriminate between the template strand and newly synthesized strand . • Newly synthesized strand is distinguished because it has not been methylated. • When the new strand containing mismatch is identified, an exonuclease removes mismatched bases.
  32. 32. • The gap left by removal of the mismatched nucleotides is filled by using DNA polymerase I. • A defect in mismatch repair in human has been identified to cause Hereditary Nonpolyposis Colon Cancer (HNPCC). • HNPCC is one of the most common inherited diseases; it affects one in 200 people and is responsible for about 15% of all colorectal cancers in the United States. • The relationship between HNPCC and defects in mismatch repair was discovered in 1993.
  33. 33. Thank You!!