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REPLICATION

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REPLICATION

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REPLICATION

  1. 1.  DNA carries genetic information from generation to generation.  Responsible to preserve the identity of the species over millions of years.  DNA may be regarded as Reserve bank of genetic information or memory bank.
  2. 2.  Some viruses contain RNA as the genetic material  DNA is more stable than RNA.  DNA is more suitable molecule for long-term repository of genetic information.
  3. 3.  The biological information flows from DNA to RNA, & from there to proteins.  This is central dogma of life.  DNA in a cell must be duplicated (replicated), maintained & passed down accurately to the daughter cells. DNA RNA Protein Replication Transcription Translation
  4. 4.  DNA is the genetic material.  When the cell divides, the daughter cells receive an identical copy of genetic information from the parent cell.  Definition:  Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA with high fidelity.
  5. 5.  Replication is semiconservative:  The parent DNA has two strands complementary to each other.  Both the strands undergo simultaneous replication to produce two daughter molecules.
  6. 6.  Each one of the newly synthesized DNA has one-half of the parental DNA (one strand from original) & one half of new DNA.  This is known as semiconservative replication - “half of the original DNA is conserved in the daughter DNA”.  Experimental evidence was provided by Meselson & Stahl (1958)
  7. 7.  The initiation of DNA synthesis occurs at a site called origin of replication.  In prokaryotes, only one site, where as in eukaryotes, there are multiple sites of origin.  These sites mostly consist of a short sequence of A-T base pairs.
  8. 8.  A specific protein called dna A(20-50 monomers) binds with the site of origin for replication.  This causes the double-stranded DNA to separate.
  9. 9.  Two complementary strands of DNA separate at the site of replication to form a bubble.  Multiple replication bubbles are in eukaryotic DNA molecules, which is essential for a rapid replication process.
  10. 10.  For the synthesis of new DNA, a short fragment of RNA (5-50 nucleotides, variable with species) is required as a primer.  The enzyme primase (a specific RNA polymerase) in association with single- stranded binding proteins (SSBP) forms a complex called primosome & produces RNA primers.
  11. 11.  A constant synthesis & supply of RNA primers should occur on the lagging strand of DNA  On leading strand only one RNA primer is required.
  12. 12.  The replication of DNA occurs in 5' to 3' direction, simultaneously, on both strands of DNA.  Leading strand (continuous or forward):  The DNA synthesis is continuous.
  13. 13.  Lagging strand (discontinuous or retrograde):  The DNA synthesis is discontinuous, short pieces of DNA (15-250 nucleotides) are produced on lagging strand.  Replication occurs in both direction from replication bubble.
  14. 14.  The separation of two strands of parent DNA results in the formation of replication fork.  The active synthesis of DNA occurs in this region.  The replication fork moves along the parent DNA as the daughter DNA molecules are synthesized.
  15. 15.  DNA helicases bind to both the DNA strands at the replication fork.  Helicases move along the DNA helix & separate the strands.  Their function is comparable with a zip opener.  Helicases are dependent on ATP for energy supply.
  16. 16.  Also called helix-destabilizing proteins.  SSB proteins bind only to single-stranded DNA.  They bind cooperatively the binding of one molecule of SSB protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand.
  17. 17.  These are not enzymes.  These will provide single-stranded template required by polymerases & also protects the DNA from nucleases that degrades single- stranded DNA.
  18. 18.  The DNA polymerases responsible for copying the DNA templates are only able to "read" the parental nucleotide sequences in the 3' to 5' direction & they synthesize the new DNA strands in the 5' to 3' (anti parallel) direction.  The two newly synthesized nucleotide chains must grow in opposite in the directions one in the 5' to 3' direction toward the replication fork & one in the 5' to 3' direction away from the replication fork.
  19. 19.  Leading strand:  The strand that is being copied in the direction of the advancing replication fork is called the leading strand & is synthesized continuously.  Lagging strand:  The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork.
  20. 20.  These short stretches of discontinuous DNA, termed Okazaki fragments & are joined to become a single, continuous strand.  This is called as lagging strand.
  21. 21.  Synthesis of a new DNA strand, catalysed by DNA polymerase lll, occurs in 5'-3' direction.  This is antiparallel to the parent template DNA strand.  The presence of all the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP & dTTP) is an essential prerequisite for replication to take place.
  22. 22.  The synthesis of two new DNA strands, simultaneously, takes place in the opposite direction - one is in a direction (5'-3') towards the replication fork which is continuous (Leading strand)  The other in a direction (5'- 3') away from the replication fork which is discontinuous (Lagging strand).
  23. 23.  The incoming deoxyribonucleotides are added one after another, to 3' end of the growing DNA chain.  A molecule of pyrophosphate (PPi) is removed with the addition of each nucleotide.  The template DNA strand (the parent) determines the base sequence of the newly synthesized complementary DNA.
  24. 24.  Prokaryotic & eukaryotic DNA polymerases elongate a new DNA strand by adding deoxy ribonucleotides, one at a time, to the 3'-end of the growing chain.  The sequence of nucleotides that are added is dictated by the base sequence of the template strand, with which the incoming nucleotides are paired.
  25. 25.  The DNA strand (leading strand) with its 3'- end (3'-OH) oriented towards the fork can be elongated by sequential addition of new nucleotides.  The other DNA strand (lagging strand) with 5'- end presents some problem,
  26. 26.  There is no DNA polymerase enzyme (in any organism) that can catalyse the addition of nucleotides to the 5‘ end (3'- 5' direction) of the growing chain.  This problem is solved by synthesizing this strand as a series of small fragments.  These pieces are made in the normal 5'-3' direction & later joined together.
  27. 27.  The small fragments of the discontinuously synthesized DNA are called Okazaki pieces.  These are produced on the lagging strand of the parent DNA.  Okazaki pieces are later joined to form a continuous strand of DNA.  DNA polymerase I & DNA ligase are responsible for this process.
  28. 28.  Fidelity of replication is the most important for the very existence of an organism.  Besides its 5'-3' directed catalytic function, DNA polymerase III also has a proof-reading activity.
  29. 29.  It checks the incoming nucleotides & allows only the correctly matched bases (i.e. complementary bases) to be added to the growing DNA strand.  DNA polymerase edits its mistakes (if any) & removes the wrongly placed nucleotide bases.
  30. 30.  For example, if the template base is cytosine & the enzyme mistakenly inserts an adenine instead a guanine into the new chain, the 3' to 5' exonuclease removes the misplaced nucleotide.  The 5' to 3' polymerase replaces it with the correct nucleotide containing guanine.
  31. 31.  The synthesis of new DNA strand continues till it is in close proximity to RNA primer.  DNA polymerase I removes the RNA primer & takes its position.  DNA polymerase I catalyses the synthesis (5'- 3' direction) of a fragment of DNA that replaces RNA primer.
  32. 32.  The enzyme DNA ligase catalyses the formation of a phosphodiester linkage between the DNA synthesized by DNA polymerase III & the small fragments of DNA produced by DNA polymerase l.  This process-nick sealing-requires energy, provided by the breakdown of ATP.  DNA polymerase II participates in the DNA repair process.
  33. 33.  The double helix of DNA separates from one side & replication proceeds, supercoils are formed at the other side.  The problem of supercoils in DNA replication is solved by a group of enzymes called DNA topoisomerases.
  34. 34.  Reversibly cut a single strand of the double helix.  They have both nuclease (strand-cutting) & ligase (strand-resealing) activities.  They do not require ATP, but rather appear to store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand.
  35. 35.  Bind tightly to the DNA double helix & make transient breaks in both strands.  The enzyme then causes a second stretch of the DNA double helix to pass through the break & finally reseals the break.  Supercoils can be relieved.
  36. 36.  Replication of DNA in eukaryotes closely resembles that of prokaryotes.  Certain differences exist.  Multiple origins of replication is a characteristic feature of eukaryotic cell.  Five distinct DNA polymerases are known in eukaryotes.
  37. 37.  DNA polymerase α is responsible for the synthesis of RNA primer for both the leading & lagging strands of DNA.  DNA polymerase β is involved in the repair of DNA.  Its function is comparable with DNA polymerasIe found in prokaryotes.
  38. 38.  DNA polymerase γ participates in the replication of mitochondrial DNA.  DNA polymerase δ is responsible for the replication on the leading strand of DNA.  It also possesses proof-reading activity.  DNA polymerase ε is involved in DNA synthesis on the lagging strand & proof- reading function.
  39. 39.  The events surrounding eukaryotic DNA replication & cell division (mitosis) are coordinated to produce the cell cycle.  The period preceding replication is called the G1 phase (Gap1).  DNA replication occurs during the S (synthesis) phase.
  40. 40.  Following DNA synthesis, there is another period (G2 phase, Gap2) before mitosis (M).  Cells that have stopped dividing, such as mature neurons, are said to have gone out of the cell cycle into the GO phase.
  41. 41.  Bacteria contain a specific type II topoisomerase namely gyrase.  This enzyme cuts & reseals the circular DNA (of bacteria) & thus overcomes the problem of supercoils.  Bacterial gyrase is inhibited by the antibiotics ciprofloxacin, novobiocin & nalidixic acid.
  42. 42.  Certain compounds that inhibit human topoisomerases are used as anticancer agents e.g. adriamycin, etoposide, doxorubicin.  The nucleotide analogs that inhibit DNA replication are also used as anticancer drugs e.g. 6-mercaptopurnie , 5-fluorouracil.
  43. 43.  The leading strand is completely synthesized  On lagging strand, removal of the RNA primer leaves a small gap which cannot be filled.  The daughter chromosomes will have shortened DNA molecule.  Over a period of time, chromosomes may lose certain essential genes & cell dies.
  44. 44.  Telomeres are the special structures that prevent the continuous loss of DNA at the end of the chromosomes during replication.  Protect the ends of the chromosomes & prevent the chromosomes from fusing with each other.  Human telomeres contain thousands of repeat TTAGGG sequences, which can be up to a length of 1500 bp.
  45. 45.  Telomerase is an unusual enzyme, it is composed of both protein & RNA.  In humans, RNA component is 450 nucleotides in length, & at 5'-terminal & it contains the sequence 5‘- CUAACCCUAAC-3'.  Central region of this sequence is complementary to the telomere repeat sequence 5'-TTAGGG-3'.  Telomerase RNA sequence can be used as a template for extension of telomere.
  46. 46.  Eukaryotic DNA is associated with tightly bound basic proteins, called histones.  These serve to order the DNA into basic structural units, called nucleosomes.  Nucleosomes are further arranged into increasingly more complex structures that organize & condense the long DNA molecules into chromosomes that can be segregated during cell division.
  47. 47.  Five classes of histones -H1, H2A, H2B, H3 & H4.  These small proteins are positively charged at physiologic pH & contain high content of lysine & arginine.  They form ionic bonds with negatively charged DNA.
  48. 48.  Two molecules each of H2A, H2B, H3 & H4 form the structural core of the individual nucleosome "beads.“  Around this core, a segment of the DNA double helix is wound nearly twice, forming a negatively super twisted helix.  Neighboring nucleosomes are joined by "linker" DNA approximately fifty base pairs long.
  49. 49.  Histone H1 of which there are several related species, is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads.  H1 is the most tissue-specific & species-specific of the histones.  It facilitates the packing of nucleosomes into the more compact structures.
  50. 50.  Nucleosomes can be packed more tightly to form a polynucleosome (also called a nucleofilament),  This structure assumes the shape of a coil, often referred to as a 30-nm fiber.  The fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins.  Additional levels of organization lead to the final chromosomal structure.
  51. 51.  Textbook of Biochemistry - U Satyanarayana  Textbook of Biochemistry - Lippincott’s

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