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For quite some time, scientists have been interested in chromosomes WHY???

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Presentation on theme: "For quite some time, scientists have been interested in chromosomes WHY???"— Presentation transcript:

1 For quite some time, scientists have been interested in chromosomes WHY???

2 Chromosomes They replicate prior to both mitosis and meiosis? How? They carry information for genetic traits (genotype determines phenotype). How? These are questions of function-to address these questions it seemed logical to look at the structure of chromosomes

3 Pre-1953-What did we know about chromosomes What is significant about 1953? Chromosomes made of DNA and protein Which of these molecules stored the genetic information? Most researchers favored protein. Why?

4 History-DNA or protein is the genetic material? Griffith-1928 Avery, McCloud, McCarty-1944 Hershey and Chase-1952 Conclusion-DNA was the genetic information in the chromosome To understand questions of function regarding genes-we had to know the structure of DNA

5 LE 16-2 Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Living S cells are found in blood sample Mouse healthy Mouse dies RESULTS

6 LE 16-3 Bacterial cell Phage head Tail Tail fiber DNA 100 nm

7 Figure 16.2b The Hershey-Chase experiment

8 The Race to discover the structure of DNA Watson and Crick Chargaff Pauling Wilkins and Franklin

9 Figure 16-01

10 LE 16-6 Franklin’s X-ray diffraction photograph of DNA Rosalind Franklin

11 X-ray diffraction insights 1.Double helix with a uniform width of 2nm 2.Purine and pyrimidine bases stacked.34 nm apart 3.Helix makes a turn every 3.4 nm 4.10 layers of nitrogen bases every turn of the helix

12 LE 16-UN298 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

13 The Birth of Genetics and Genetic Engineering

14 The “Double Helix” paper A copy is posted on cell web site-please read it Major insights: A. DNA is a double helix B. The two strands are held together by hydrogen bonding between complementary base pairs (A-T) and G-C) DNA is antiparallel

15 LE 16-5 Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) DNA nucleotide Phosphate 3 end Guanine (G) Sugar (deoxyribose)

16 LE 16-8a Adenine (A) Thymine (T) Sugar

17 LE 16-8b Guanine (G) Cytosine (C) Sugar

18 LE 16-7b 5 end 3 end 5 end 3 end Partial chemical structure Hydrogen bond

19 LE 16-7a Key features of DNA structure 0.34 nm 3.4 nm 1 nm

20 LE 16-7c Space-filling model

21 Structure answers a question of function Question-How does DNA replicate? “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Semi-conservative replication

22 LE 16-9_1 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

23 LE 16-9_2 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The first step in replication is separation of the two DNA strands.

24 LE 16-9_3 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

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26 Experimental Evidence for Semi- conservative Replication Just because something is logical does not mean it is true. Three possible mechanisms of DNA replication- A. Conservative Semi-conservative C. Dispersive Messelson and Stahl experiment

27 LE 16-10 Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second replication

28 LE 16-10a Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Parent cell First replication Second replication

29 LE 16-10b Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Parent cell First replication Second replication

30 LE 16-10c Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second replication

31 Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)

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33 DNA replication-It’s more complicated than Watson and Crick thought Considerations-DNA replication 1. DNA must unwind (it’s a double helix) 2. It’s fast (mammals-50 nucls/sec; bacteria-500 nucls/sec). 3.Accuracy-1 mistake/1 billion nucleotides 4. DNA polymerase limitations-can’t synthesize denovo; only works in 5’  3’ direction 5. DNA is antiparallel

34 DNA replication proteins Several of the replication considerations suggest the involvement of proteins (especially enzymes) in DNA replication

35 Consideration #1-DNA must unwind prior to replication DNA helicase (unwindase) Topoisomerase (relieves twisting) Single strand binding proteins

36 Consideration #2-Speed of Replication Enzymes involved-DNA polymerase (11 forms in eukaryotes)-III is the major replicative enzyme) DNA replication is bi-directional

37 LE 16-13 New strand 5 end Phosphate Base Sugar Template strand 3 end 5 end 3 end 5 end 3 end 5 end 3 end Nucleoside triphosphate DNA polymerase Pyrophosphate

38 LE 16-12 In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. Two daughter DNA molecules Parental (template) strand Daughter (new) strand 0.25 µm Replication fork Origin of replication Bubble In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

39 Consideration #3-Accuracy DNA polymerase has “proofreading capabilities”-mismatch repair

40 Consideration #4-Limitations of DNA polymerase DNA polymerase can’t synthesize a new strand “denovo”-needs a free 3’ OH group to attach the next nucleotide to Solution-RNA primase-adds RNA primer (5-10 nucleotides)-later primer removed by a form of DNA polymerase that replaces RNA nucleotides with DNA nucleotides Pieces of DNA joined by DNA ligase

41 LE 16-15_1 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication

42 LE 16-15_2 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

43 Consideration #4-Limitations of DNA polymerase (continued) DNA polymerase only works in 5’  3’ direction Why is this a problem? Because of consideration #5-DNA is antiparallel-One strand runs in the 5’  3’ direction; the other runs in the 3’  5’ direction Solution1- Is there a 3’  5’ DNApolymerase? (haven’t found one yet)

44 Solution 2-DNA replication occurs differently on the 2 strands Leading strand (continuous replication) Lagging strand (discontinuous replication)- involvement of Okasaki fragments (approximately 200 nucleotides in length in eukaryotes).

45 LE 16-14 Parental DNA 5 3 Leading strand 3 5 3 5 Okazaki fragments Lagging strand DNA pol III Template strand Leading strand Lagging strand DNA ligase Template strand Overall direction of replication

46 LE 16-15_1 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication

47 LE 16-15_2 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

48 LE 16-15_3 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off.

49 LE 16-15_4 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off.

50 LE 16-15_5 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 3 3 5 5 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2.

51 LE 16-15_6 5 3 Primase joins RNA nucleotides into a primer. Template strand 5 3  Overall direction of replication RNA primer 3 5 3 5 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Okazaki fragment 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 3 3 5 5 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. 3 3 5 5 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete.

52 LE 16-16 5 3 Parental DNA 3 5 Overall direction of replication DNA pol III Replication fork Leading strand DNA ligase Primase OVERVIEW Primer DNA pol III DNA pol I Lagging strand Lagging strand Leading strand Leading strand Lagging strand Origin of replication

53 Figure 16.15 The main proteins of DNA replication and their functions

54 Repair of Damaged DNA Environmental factors including UV radiation can damage DNA DNA polymerase can repair damage (excision repair)

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56 LE 16-17 DNA ligase DNA polymerase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. Repair synthesis by a DNA polymerase fills in the missing nucleotides. A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease A thymine dimer distorts the DNA molecule.


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