REPLICATION

REPLICATION

1. DNA Replication Gandham.Rajeev Email:[email protected]

2. Introduction 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.

3. 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.

4. Central Dogma of Life 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

5. Replication of DNA 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.

6. Replication in Prokaryotes Replication is semiconservative: The parent DNA has two strands complementary to each other. Both the strands undergo simultaneous replication to produce two daughter molecules.

7. 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)

8. Initiation of Replication 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.

9. 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.

10. Replication bubbles 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.

11. RNA Primer 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.

12. A constant synthesis & supply of RNA primers should occur on the lagging strand of DNA On leading strand only one RNA primer is required.

13. DNA synthesis is semidiscontinuous & bidirectional 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.

14. 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.

15. Replication fork & DNA synthesis 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.

16. DNA helicases 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.

17. Single-stranded DNA-binding (SSB) proteins 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.

18. These are not enzymes. These will provide single-stranded template required by polymerases & also protects the DNA from nucleases that degrades single-stranded DNA.

19. Direction of DNA replication 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.

20. 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.

21. These short stretches of discontinuous DNA, termed Okazaki fragments & are joined to become a single, continuous strand. This is called as lagging strand.

22. DNA Polymerase III 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.

23. 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).

24. 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.

25. Chain elongation 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.

26. Elongation of leading & lagging strands

27. Polarity problem 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,

28. 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.

29. Okazaki pieces 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.

30. Proof-reading function of DNA Polymerase III 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.

31. 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.

32. 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.

33. Polymerase & proof reading function of DNA polymerase III

34. Replacement of RNA primer by DNA 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.

35. Removal of RNA primer & gap filling by DNA polymerase I

36. 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.

37. Formation of phosphodiester bond by DNA ligase

38. Supercoils & DNA topoisomerases 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.

39. Positive supercoiling

40. Type I DNA Topoisomerases 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.

41. Type I DNA topoisomerases

42. Type II DNA topoisomerases 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.

43. Type II DNA topoisomerases

44. Replication in eukaryotes 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.

45. DNA Polymerases 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.

46. 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.

47. The eukaryotic cell cycle 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.

48. 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.

49. Cell cycle

50. Inhibitors of DNA Replication 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.

51. 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.

52. Telomeres & telomerase 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.

53. 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 .

54. 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. Role of telomerase

55. Role of telomerase

56. 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. Organization of eukaryotic DNA

57. Histones & formation of nucleosomes 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.

58. Nucleosomes 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.

59. 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.

60. Higher levels of Organization 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.

61. Structural Organization of eukaryotic DNA

62. References Textbook of Biochemistry - U Satyanarayana Textbook of Biochemistry - Lippincott’s

63. Thank you