1. DUPLICACION DEL MATERIAL GENETICO

2. The Eukaryotic Cell Cycle DNA Synthesis Restriction Point Mitosis Quiescence S S S G1 S M S G2 S G1

3. II. Historical Background A. 1953 Watson and Crick: DNA Structure Predicts a Mechanism of Replication “It has no escaped our notice that the specific pair we have postulated immediately suggests a possible copying mechanism for the genetic material.” B. 1958 Meselson and Stahl: DNA Replication is Conservative

4. The Meselson-Stahl Experiment “the most beautiful experiment in biology.” Three potential DNA replication models and their predicted outcomesThe actual data! 1/4 old: 3/4 new 1/2 hybids: 1/2 new All hybrids 1/2 old: 1/2 new All hybrids

5. III. General Features of DNA Replication 1. requires a DNA template and a primer with a 3’ OH end. (DNA synthesis cannot initiate de novo) 2. requires dNTPs. 3. occurs in a 5’ to 3’ direction. DNA Synthesis: Short RNA molecules act as primersin vivo

7. DNA replication is extremely accurate Error rates of ~1 in 10 9 to 10 10 for cellular DNA replication This would allow approximately 1 human genome to be replicated with only a few errors!! How can this happen if the intrinsic error rate of the best polymerases is only ~1 per 10 4 to 10 5 nucleotides? Proofreading – additional 10 2 to 10 3 -fold increased fidelity Uses 3´ to 5´ exonuclease activity Mismatch repair – final 10 2 to 10 3 -fold increased fidelity

9. Model for the Interaction of Klenow Fragment with DNA

10. How the Proofreading Activity of Klenow Fragment Works

12. Aplicación de la Polimerización Traslado del Corte o “Nick Translation”

14. DNA Polymerase I can Perform “Nick Translation” They act together to edit out sections of damaged DNA

18. Procesividad de la Duplicación del DNA

22. DNA replication is highly processive Pol III holoenzyme of E. coli can synthesize hundreds of thousands of nucleotides before falling off the template. Processivity is effected by the beta subunit of the polymerase, called the sliding clamp.

24. Replication in Eukaryotes Replication in eukaryotes (~50 nucleotides/sec) is much slower than in prokaryotes (~1,000 nucleotides/sec). FunctionE. coliHuman Genomic replicationpol IIIpol delta Primer synthesis (RNA/DNA)Primasepol alpha Sliding clamp beta-subunit of pol III proliferating cell nuclear antigen (PCNA) PCNA originally discovered in sera of patients with the autoimmune disorder, SLE (systemic lupus erythematosis). It is a highly-regulated marker of cell proliferation.

25. The problem of replication of the ends of linear chromosomes 3´5´3´5´ 3´3´ DNA replication cannot complete the 3´ end of linear chromosomes The cell addresses this issue by generating hundreds to thousands of simple repeats (5´TTAGGG)n at the ends of chromosomes of all vertebrates - telomeres The enzyme, telomerase, is an RNA-directed DNA polymerase.

26. DNA Pol I y DNA Pol III trabajan juntas

27. DNA Pol I RNA Okazaki fragment >10 kb 1 kb Roles of DNA Pol III and Pol I in E. coli Pol III—main DNA replication enzyme. It exists as a dimer to coordinate the synthesis of both the leading and lagging strands at the replication fork. Pol I—repair enzyme to remove RNA primers that initiate DNA synthesis on both strands. It is need predominantly for maturation of Okazaki fragments. 1) Removes RNA primers (5’  3’ Exo) 2) Replaces the RNA primers with DNA (5’  3’ Pol & 3’  5’ Exo proofreading) RNA primer replaced with DNA by Pol I’s nick translatiton activity Okazaki fragment

29. Dirección de la Replicación

30. Initiation of replication Prokaryotic and eukaryotic cellular replication Some viruses In higher eukaryotes, number and characteristics of origins are not well defined. Origin activation is extremely complex, and involves both sequence (cis) elements and protein (trans) elements.

32. Replication of the E. coli Chromosome is Bidirectional

35. DNA mitocondrial Un ejemplo de replicación alternativa

36. Mammalian Mitochondrial DNA (MtDNA) Multi-copy, circular molecule of ~16,000 bp. 2. Encodes genes for respiration (13 proteins) and translation (22 tRNAs, 2 rRNAs). 3. 2 promoters (1 on each strand); the STOP codons for the protein genes, UAA, created post-transcriptionally by polyadenylation 4. Some genetic diseases caused by mutations in mtDNA. MtDNA mutations accumulate during aging. 5. MtDNA used to define phylogenetic relationships between species, subspecies, etc., or define breeding populations.

37. Mammalian Mt DNA

38. Mt DNA replication

39. Mammalian (mouse) mtDNA Replication Two origins of replication: H (for heavy strand) and L (for light strand) that are used sequentially for unidirectional replication. Persistent D-loop at H ori, which is extended to start replication of the H strand. Once ~2/3 of H strand is replicated, L ori is exposed and replication of L strand starts. The lagging L strand replication gives 2 type of molecules:  and  is gapped on L strand.  L strand finishes replicating, and then both  and  are converted to supercoiled forms.

40. En la replicación del DNA participan otras enzimas además de las DNA polimerasas

42. DNA replication is semi-discontinuous Lagging strand synthesis MUST be semi-discontinuous

43. Functional aspects of DNA replication FunctionProteins Unwind helixDNA helicases Relieve torsional stressTopoisomerase DNA polymerizationDNA polymerase Primer (RNA) synthesisPrimase Elimination of RNA primers5´-3´ exonuclease Proofreading3´-5´ exonuclease Joining DNA strands following DNA ligase primer elimination Protect local single-strand regionsSingle-strand binding proteins

44. Replication of the E. coli Chromosome is Semidiscontinuous Replicates continuously DNA synthesis is going in same direction as replication fork Because of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in the direction of fork movement in both the 5’ to 3’ and 3’ to 5’ directions overall. Replicates discontinuously DNA synthesis is going in opposite direction as replication fork However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only. The solution is semidiscontinuous DNA replication. Joined by DNA ligase

45. Review of DNA synthesis – E. coli as paradigm

46. At Each Replication Fork is A Replisome

47. LAS TOPOISOMERASAS

48. Additional Terms Used To Describe Topology The Linking Number Difference =  L = L – L 0 It is a measure of the number of writhes For a relaxed molecule:  L = 0 The difference between the linking number of a DNA molecule (L) and the linking number of its relaxed form (L 0 ) The superhelical density (  )=  L – L 0 It is a measure of supercoiling that is independent of length. For a relaxed molecule:  = 0 DNA in cells has a  of –0.06

49. What Topoisomerases Do 1. Change the linking number of a DNA molecule by: A) Breaking one or both strands then B) Winding them tighter or looser, and rejoining the ends. 2. Usually relax supercoiled DNA

50. Type I Topoisomerases Topo I from E. coli 1) acts to relax only negative supercoils 2) increases linking number by +1 increments Topo I from eukaryotes 1) acts to relax positive or negative supercoils 2) changes linking number by –1 or +1 increments

51. Maximum supercoiled 3 min. Topo I 25 min. Topo I Relaxation of SV40 DNA by Topo I

52. Type II Topoisomerases All Type II Topoisomerases Can Catenate and Decatenate cccDNA molecules Circular DNA molecules that use type II topoisomerases: E. coli Eukaryotes -plasmids-mitochondrial DNA -E. coli chromosome-circular dsDNA viruses (SV40)

53. An E. coli Type II Topoisomerase: DNA Gyrase Topo II (DNA Gyrase) from E. coli 1) Acts on both neg. and pos. supercoiled DNA 2) Increases the # of neg. supercoils by increments of 2 3) Requires ATP

54. DNA Gyrase Adds Negative Supercoils to DNA

55. Topo II from Eukaryotes 1) Relaxes only negatively supercoiled DNA 2) Increases the linking number by increments of +2 3) Requires ATP

56. The Role of Topoisomerases in DNA Replication DNA gyrase Example 1: DNA gyrase (a type II topo of E. coli removes positive supercoils that normally form ahead of the growing replication fork

57. Example 2: Replicated circular DNA molecules are separated by type II topoisomerase

58. A Review of the Different Topoisomerases +1 or –1 supercoils Cleaves 1 strand (nicks) Cleaves 2 strands (ds cut) Can catenate and decatenate DNA

59. How Does Eukaryotic DNA Become Neg. Supecoiled? Plectonemic Toroidal (Solenoidal) Q: What happens when you remove the histone core?A: The negative supercoil adopts a plectonemic conformation

60. Aplicación del conocimiento de las Topoisomerasas

61. At Each Replication Fork is A Replisome

62. different agents used in Bacterial infection or cancer chemotherapy Targeting DNA Replication: Topoisomerase Inhibitors

63. nick DNA, pass other strand through nick ATP-independent; change linking number in steps of 1 Inhibitors (e.g., camptothecin) can freeze enzyme-DNA covalent complex Type I Topoisomerase

64. break DS DNA, pass DS DNA through enzyme-bound nick require ATP; change linking number in steps of 2 bacterial DNA gyrase uses ATP to increase linking number Type II Topoisomerases

65. Nalidixic acid 1 2 3 6 4 7 5 Quinolones and fluoroquinolones bind to two enzymes needed for bacterial replication, DNA gyrase (A subunit mainly) and topoisomerase IV, causing inhibition of DNA replication and cell death. Mammalian homologues show 100-1000 times less affinity for these drugs. Cinoxacin Resistance developed due to gyrase mutations. Nalidixic acid and cinoxacin are well absorbed from GI tract and rapidly metabolized in the liver (one metabolite, OH-nalidixic acid is active). They only reach effective concentration in urine. Early Quinolones Used for UTI

66. Fluoroquinolones are active against most urinary tract pathogens: E. coli and Klebsiella. Also most bacteria that cause enteritis: Salmonella, Shigella, E. coli. Inactive against anaerobes: Clostridium difficile Rapidly and incompletely absorbed from the GI tract. Widely distributed to body fluids but concentrations in CSF are low. Plasma lifetime varies from 4-11 hours. lomefloxacin ciprofloxacin norfloxacin ofloxacin Ciprofloxacin reaches high concentration in respiratory, urinary and GI tract, bones, joints, skin, and soft tissues. It is eliminated mostly by renal clearance. Newer derivatives Grepafloxacin, Levofloxacin, Gatifloxacin, Clinafloxacin Moxifloxacin, Trovafloxacin can have increased activity against gram (+) and anaerobic bacteria, but are not generally first line drugs for these organisms. Fluoroquinolones

67. Fluoroquinolone resistance mutations: DNA gyrase is the primary target in E. coli and other gram-negative organisms topoisomerase IV is primary target for S. aureus and other gram-positive bacteria.

68. Patología por falla de Helicasa

69. Sindrome de Werner

70. Genes implicated in progerias: Werner’s:  found gene implicated in Werner’s  Werner’s gene appears to be responsible for making a protein helicase is responsible for unwinding dsDNA The genetic sequence of Werner’s gene closely resembles helicases a sequence of genes that code for helicases in normal cells

71. DNA Replication Mutations of helicases may affect unwinding of DNA Could affect following: - DNA repair - DNA replication - gene expression - chromosome recombination recombination

72. Aging Hypothesis: # of defects  With  age there are a # of defects in genes that code for helicases in the cell abnormal proteins  This produces abnormal proteins that can’t unwind ds DNA  Result in a  in the efficiency of above cellular functions  Ultimately leads to a  in functional capacity.

74. Quimioterapia Anti-viral basado en el conocimiento de la replicación

76. Viral enzymes Nucleic acid polymerases DNA-dependent DNA polymerase - DNA viruses RNA-dependent RNA polymerase - RNA viruses RNA dependent DNA polymerase (RT) - Retroviruses Protease (retrovirus) Integrase (retrovirus) Neuraminidase (orthomyxovirus) Anti-Viral Chemotherapy

77. 1962 Idoxuridine Pyrimidine analog Toxic Topical - Epithelial herpetic keratitis 1983 Acyclovir Purine analog Sugar modification Chain terminator Anti-herpes Selective to virus-infected cells 1990’s Protease inhibitors Anti-Viral Chemotherapy

79. Nucleic Acid Synthesis Polymerases are often virally encoded Other enzymes in nucleic acid synthesis e.g. THYMIDINE KINASE in Herpes Simplex Anti-Viral Chemotherapy

80. Thymidine Kinase Deoxy-thymidine Deoxy-thymidine triphosphate Intracellular viral or cellular thymidine kinase adds first phosphate PO 4 Cellular kinases add two more phosphates to form TTP Anti-Viral Chemotherapy

81. Why does Herpes simplex code for its own thymidine kinase? TK- virus cannot grow in neural cells because they are not proliferating (not making DNA) Although purine/pyrimidines are present, levels of phosphorylated nucleosides are low Allows virus to grow in cells that are not making DNA “Thymidine kinase” is a misnomer Deoxynucleoside kinase NON-SPECIFIC Anti-Viral Chemotherapy

82. Herpes thymidine kinase will phosphorylate any deoxynucleoside including drugs – as a result of its necessary non-specificity Nucleoside analog may be given in non-phosphorylated form Gets drugs across membrane Allows selectivity as only infected cell has enzyme to phosphorylate the drug ACG P PP Anti-Viral Chemotherapy Cellular TK (where expressed) does not phosphorylate (activate) the drug

83. Need for activation restricts drug to: Viruses such as HSV that code for own thymidine kinase Virus such as cytomegalovirus and Epstein-Barr virus that induce cells to overproduce their own thymidine kinase In either case it is the VIRUS-INFECTED cell that activates the drug Anti-Viral Chemotherapy

84. Thymidine kinase activates drug but phosphorylated drug inhibits the polymerase Nucleotide analogs Sugar modifications Base modifications Selectivity Viral thymidine kinase better activator Cellular enzyme may not be present in non-proliferating cells Activated drug is more active against viral DNA polymerase that against cell polymerase Anti-Viral Chemotherapy

85. Guanine analogs Acyclovir = acycloguanosine = Zovirax Ganciclovir = Cytovene Activated by viral TK Activated ACV is better (10x) inhibitor of viral DNA polymerase than inhibitor of cell DNA polymerase Excellent anti-herpes drug Acyclovir Ganciclovir Anti-Viral Chemotherapy

86. Acyclovir: Chain terminator Good anti-herpes drug T PP G P C P A Normal DNA synthesis Anti-Viral Chemotherapy

87. T PP G P C P A P A ACG P-P-P Terminatio n Also inhibits: Epstein Barr Cytomegalovirus Acyclovir: Chain terminator Selective: Virus phosphorylates drug Polymerase more sensitive Anti-Viral Chemotherapy

88. Acyclovir very effective against: Herpes simplex keratitis (topical) Latent HSV (iv) Fever blisters – Herpes labialis (topical) Genital herpes (topical, oral, iv) Resistant mutants in thymidine kinase or DNA polymerase Appears not to be teratogenic or carcinogenic Ganciclovir very effective against cytomegalovirus – viral DNA polymerase is very sensitive to drug activated by cell TK Anti-Viral Chemotherapy

89. Adenine arabinoside (Ara-A) Problems : Severe side effects Resistant mutants (altered polymerase) Chromosome breaks (mutagenic) Tumorigenic in rats Teratogenic in rabbits Insoluble Use: topical applications in ocular herpes simplex Competitive inhibitor of virus DNA polymerase which is much more sensitive than host polymerase Anti-Viral Chemotherapy

90. Adenine arabinoside HSV encephalitis Neonatal herpes Disseminated herpes zoster Hepatitis B Poor in vivo efficacy: DEAMINATION Anti-Viral Chemotherapy

91. Other sugar modifications: AZT azidothymidin e DDI dideoxyinosi ne DDC dideoxycytidi ne Anti-Viral Chemotherapy

92. Base change analogs Altered base pairing Mutant DNA Resistant mutants Trifluorouridine Viroptic anti-HSV Idoxuridine Anti-Viral Chemotherapy

93. Fluoroiodo aracytosine has both a base and a sugar alteration O HOCH 2 O NH 2 I F Anti-Viral Chemotherapy

94. Prodrugs e.g. Famciclovir Taken orallyConverted by patient’s metabolism HSV thymidine kinase P Host kinase P P Penciclovir: Available as topical cream Glaxo-SmithKlein

95. Non-nucleoside Non-competitive RT inhibitors Combination therapy with AZT Resistance mutations will be at different sites The most potent and selective RT inhibitors Nanomolar range Minimal toxicity (T.I. 10,000-100,000) Synergistic with nucleoside analogs (AZT) Good bio-availability Resistant mutants - little use in monotherapy Anti-Viral Chemotherapy

96. Sustiva (S) -6- chloro-4- (cyclopropylethynyl)-1,4-dihydro-4- (trifluoromethyl)-2H-3, 1- benzoxazin-2-one. DuPont Anti-Viral Chemotherapy

97. Nevirapine: Approved for AIDS patients Good blocker of mother to child transmission peri-natal - breast feeding Single dose at delivery reduced HIV transmission by 50% Single dose to baby by 72 hours Efavirenz (Sustiva, DMP266) In combination therapy will suppress viral load as well as HAART and may be better – Approved for AIDS patients Anti-Viral Chemotherapy

98. Phosphono acetic acid (PAA) Phosphono formic acid O O HO P C OH Binds pyrophosphate site of polymerase Competitive inhibitor 10 -100x greater inhibition of herpes polymerase Toxic: accumulates in bones, nephrotoxicity Rapid resistance Clinical trial: CMV in AIDS patients Anti-Viral Chemotherapy

99. Ribavirin Guanosine analog Non-competitive inhibitor of RNA polymerase in vitro Little effect on ‘flu in vitro Often good in animals but poor in humans Aerosol use: respiratory syncytial virus i.v./oral: reduces mortality in Lassa fever, Korean and Argentine hemorrhagic fever Anti-Viral Chemotherapy