DNA AND IT S ROLE IN HEREDITY

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1 DNA AND IT S ROLE IN HEREDITY

2 Lesson overview and objectives - DNA/RNA structural properties What are DNA and RNA made of What are the structural differences between DNA and RNA What is the structure of DNA and how was it determined - DNA replication - How is DNA replicated - What mechanisms are in place to deal with errors? - DNA as the transporter of information (heredity) 2 CBS, Department of Systems Biology

3 DNA: BASIC FEATURES 3 CBS, Department of Systems Biology

4 DNA and RNA are based on sugars Five-carbon sugars (pentoses) 5 4 C C H H 2 OH O H H C 3 2 OH Ribose C OH OH C H C C H H 2 OH O H H C C 3 2 OH H Deoxyribose OH C H 1 Ribose and deoxyribose each have five carbons, but very different chemical properties and biological roles. 4 CBS, Department of Systems Biology

5 The sugar is bound to a base to form a nucleoside 5 CBS, Department of Systems Biology

6 The nucleoside binds to phosphate groups to form nucleotides 6 CBS, Department of Systems Biology

7 Both RNA and DNA form polymers 7 CBS, Department of Systems Biology

8 Both RNA and DNA can have complex 3D structures 8 CBS, Department of Systems Biology

9 DNA STRUCTURE 9 CBS, Department of Systems Biology

10 A picture that changed a lot of things 10 CBS, Department of Systems Biology

11 Rosalind Franklin and the helix 11 CBS, Department of Systems Biology

12 Chargaff Rule In 1950 Erwin Chargaff found in the DNA from many different species: Amount of A = amount of T Amount of C = amount of G Or, the abundance of purines = the abundance of pyrimidines. 12 CBS, Department of Systems Biology

13 Chargaff Rule 13 CBS, Department of Systems Biology

14 What Is the Structure of DNA? - Franklin s X-ray crystallography convinced them the molecule was helical. - Other evidence suggested there were two polynucleotide chains that ran in opposite directions antiparallel. - In 1953, Watson and Crick established the general structure of DNA. 14 CBS, Department of Systems Biology

15 Figure 13.8 DNA Is a Double Helix (Part 1) 15 CBS, Department of Systems Biology

16 Figure 13.8 DNA Is a Double Helix (Part 2) 16 CBS, Department of Systems Biology

17 What Is the Structure of DNA? Four key features of DNA structure: It is a double-stranded helix of uniform diameter It is right-handed It is antiparallel Outer edges of nitrogenous bases are exposed in the major and minor grooves 17 CBS, Department of Systems Biology

18 What Is the Structure of DNA? Complementary base pairing: Adenine (A) pairs with thymine (T) by two hydrogen bonds Cytosine (C) pairs with guanine (G) by three hydrogen bonds Every base pair consists of one purine and one pyrimidine 18 CBS, Department of Systems Biology

19 19 CBS, Department of Systems Biology

20 DNA REPLICATION 20 CBS, Department of Systems Biology

21 21 CBS, Department of Systems Biology

22 How Is DNA Replicated? Two steps in DNA replication: The double helix is unwound, making two template strands New nucleotides are added to the new strand at the 3 end and joined by phosphodiester linkages. Sequence is determined by complementary base pairing 22 CBS, Department of Systems Biology

23 Figure Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 1) 23 CBS, Department of Systems Biology

24 Figure Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 2) 24 CBS, Department of Systems Biology

25 Figure DNA Polymerase Binds to the Template Strand (Part 2) 25 CBS, Department of Systems Biology

26 How Is DNA Replicated? The machinery DNA polymerases work very fast: They are processive: Catalyze many polymerizations each time they bind to DNA Newly replicated strand is stabilized by a sliding DNA clamp (a protein) The sliding DNA clamp was recognized in dividing cells called the proliferating cell nuclear antigen (PCNA). PCNA also helps to orient the polymerase for substrate binding, binds other proteins, and is involved in other processes. 26 CBS, Department of Systems Biology

27 Figure A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization 27 CBS, Department of Systems Biology

28 28 CBS, Department of Systems Biology

29 How Is DNA Replicated? The Lagging strand This works great and easy on the leading strand. In this direction, DNA Pol III can work on a 5 ->3 direction following the forward movement of the replication fork. Now, what happens with the other strand, the lagging strand? Here, the natural direction of DNA synthesis would be 3 ->5 unfortunately, DNA Pol III can only work 5 ->3. Therefore, replication on lagging strand has to be done in another way 29 CBS, Department of Systems Biology

30 Direction of synthesis Direction of overall DNA replication 30 CBS, Department of Systems Biology

31 Direction of synthesis Direction of overall DNA replication 31 CBS, Department of Systems Biology

32 Direction of synthesis Direction of overall DNA replication 32 CBS, Department of Systems Biology

33 Direction of synthesis Direction of overall DNA replication 33 CBS, Department of Systems Biology

34 How is DNA replicated: the players DNA Helicase: unwinds the DNA molecule ssdna-binding proteins: stabilize the open molecules Primase: synthesizes an RNA primer onto the exposed strands to start DNA replication PCNA (proliferating cell nuclear antigen): stabilises, directs and guides the replication machinery DNA Polymerase III: extends the growing DNA strand in a 5 ->3 strand DNA Polymerase I: replaces RNA primer with DNA DNA Ligase: closes the gap between growing molecules 34 CBS, Department of Systems Biology

35 How Is DNA Replicated? Recap Switch to animation! J 35 CBS, Department of Systems Biology

36 How Is DNA Replicated? Origin of replication - Small, circular chromosomes have a single origin of replication. - As DNA moves through the replication complex, two interlocking circular chromosomes are formed. - DNA topoisomerase separates the two chromosomes. - Large linear chromosomes have many hundreds of origins of replication. - Replication complexes bind to the sites at the same time and catalyze simultaneous replication. 36 CBS, Department of Systems Biology

37 37 CBS, Department of Systems Biology

38 How Is DNA Replicated? Telomeres 38 CBS, Department of Systems Biology

39 How Is DNA Replicated? Telomeres Eukaryote chromosomes have repetitive sequences at the ends called telomeres. These repeats are protective and prolong cell division, especially in rapidly-dividing cells, like bone marrow. Telomerase contains an RNA sequence acts as template for telomeric DNA sequences. 39 CBS, Department of Systems Biology

40 40 CBS, Department of Systems Biology

41 How Is DNA Copied? Three possible replication patterns: Semiconservative: Parent serves as a template and new molecules have one old and one new strand Conservative: Original helix only serves as a template Dispersive: Parent fragments serve as templates, assembling old and new parts into molecules 41 CBS, Department of Systems Biology

42 42 CBS, Department of Systems Biology

43 How Is DNA copied? - Meselson and Stahl showed that semiconservative replication was the correct model. - They used density labeling to distinguish parent DNA strands from new DNA strands. - DNA was labeled with 15 N, making it more dense. 43 CBS, Department of Systems Biology

44 Figure The Meselson Stahl Experiment (Part 1) 44 CBS, Department of Systems Biology

45 Figure The Meselson Stahl Experiment (Part 2) 45 CBS, Department of Systems Biology

46 13.3 How Is DNA copied? - Results of their experiment explained by the semi-conservative model: - If conservative, the first generation of individuals would have been both high and low density, but not intermediate. - If dispersive, density in the first generation would be half, but this density would not appear in subsequent generations. 46 CBS, Department of Systems Biology

47 How Are Errors in DNA Repaired? DNA polymerases make mistakes in replication, and DNA can be damaged in living cells. Cells have three repair mechanisms: Proofreading Mismatch repair Excision repair 47 CBS, Department of Systems Biology

48 48 CBS, Department of Systems Biology

49 DNA IN HEREDITY 49 CBS, Department of Systems Biology

50 What Is the Evidence that the Gene Is DNA? By the 1920s, it was known that chromosomes consisted of DNA and proteins. A new dye stained DNA and provided circumstantial evidence that DNA was the genetic material: It was in the right place It varied among species It was present in the right amount Frederick Griffith, working with two strains of Streptococcus pneumoniae determined that a transforming principle from dead cells of one strain produced a heritable change in the other strain. 50 CBS, Department of Systems Biology

51 Figure 13.1 Genetic Transformation 51 CBS, Department of Systems Biology

52 What Is the Evidence that the Gene Is DNA? To identify the transforming principle: Oswald Avery treated samples to destroy different molecules; if DNA was destroyed, the transforming activity was lost. There was no loss of activity with destruction of proteins, carbohydrates, or lipids. 52 CBS, Department of Systems Biology

53 Figure 13.2 Genetic Transformation by DNA (Part 1) 53 CBS, Department of Systems Biology

54 Figure 13.2 Genetic Transformation by DNA (Part 2) 54 CBS, Department of Systems Biology

55 What Is the Evidence that the Gene Is DNA? Hershey-Chase experiment: Used bacteriophage T2 virus to determine whether DNA, or protein, is the genetic material Bacteriophage proteins were labeled with 35 S; the DNA was labeled with 32 P 55 CBS, Department of Systems Biology

56 Figure 13.3 Bacteriophage T2: Reproduction Cycle 56 CBS, Department of Systems Biology

57 Figure 13.4 The Hershey Chase Experiment (Part 1) 57 CBS, Department of Systems Biology

58 What Is the Evidence that the Gene Is DNA? - Genetic transformation of eukaryotic cells, transfection, can be demonstrated. - A genetic marker a gene that confers an observable phenotype is used. - Any cell can be transfected, even an egg cell result is a transgenic organism. 58 CBS, Department of Systems Biology

59 Figure 13.5 Transfection in Eukaryotic Cells (Part 1) 59 CBS, Department of Systems Biology

60 DNA: FINAL POINTS 60 CBS, Department of Systems Biology

61 SUMMARY DNA is a double-stranded helix, with the two strands connected by hydrogen bonds. A bases are always paired with Ts, and Cs are always paired with Gs, which is consistent with and accounts for Chargaff's rule. Most DNA double helices are right-handed; that is, if you were to hold your right hand out, with your thumb pointed up and your fingers curled around your thumb, your thumb would represent the axis of the helix and your fingers would represent the sugar-phosphate backbone. Only one type of DNA, called Z-DNA, is left-handed. The DNA double helix is anti-parallel, which means that the 5' end of one strand is paired with the 3' end of its complementary strand (and vice versa). As shown in Figure 4, nucleotides are linked to each other by their phosphate groups, which bind the 3' end of one sugar to the 5' end of the next sugar. Not only are the DNA base pairs connected via hydrogen bonding, but the outer edges of the nitrogen-containing bases are exposed and available for potential hydrogen bonding as well. These hydrogen bonds provide easy access to the DNA for other molecules, including the proteins that play vital roles in the replication and expression of DNA 61 CBS, Department of Systems Biology

62 DNA and the Dogma DNA has four important functions double-helical structure is essential: Genetic material stores genetic information millions of nucleotides; base sequence encodes huge amounts of information. Genetic material is susceptible to mutation a change in information possibly a simple alteration to a sequence Genetic material is precisely replicated in cell division by complementary base pairing. Genetic material is expressed as the phenotype nucleotide sequence determines sequence of amino acids in proteins. 62 CBS, Department of Systems Biology

63 DNA and the Dogma Before getting into DNA replication, it is important to state the role of DNA in life and, to be more precise, to state what is meant with The Dogma of molecular biology : - DNA can replicate itself exactly (Replication) - DNA can copy its information into RNA (Transcription) - RNA sequence is translated into protein (Translation) 63 CBS, Department of Systems Biology

64 DNA and the Dogma 64 CBS, Department of Systems Biology

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