Human Genetics: concepts and applications 5th edition Ricki Lewis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chapter 9 DNA Structure and Replication

9-2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A history of DNA Friedrich Miescher, 1871 isolated nuclei from pus and identified nuclein later called nucleic acid. Archibald Garrod, 1902 linked inheritance of “inborn errors of metabolism” with the lack of particular enzyme proteins

9-3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Discovery of a “transforming principle” Frederick Griffith, 1928 Pneumonia (Diplococcus pneumoniae) infects mice. Mice develop pneumonia and die. Two types of bacteria: R bacteria rough coat - no pneumonia S bacteria smooth coat- pneumonia Coat type is associated with virulence.

9-4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Injection Bacterial colonies Rough nonvirulent (strain R) Results Mouse healthy Smooth virulent (strain S) Mouse dies Heat-killed smooth virulent (strain S) Live strain S bacteria in blood sample from dead mouse Mouse dies Mouse healthy + Rough nonvirulent (strain R) Heat-killed smooth virulent (strain S) Griffith’s experiment identifying the “transforming principle”

9-5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. What is the “transforming principle”? Conclusion: DNA is the transforming principle allowing R bacteria to make a smooth coat and allow infection. Oswald Avery, Colin MacLeod and Maclyn McCarty, 1944 Heat-killed S bacteria “transformed” the R bacteria to a form that causes pneumonia

9-6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA is the genetic material Alfred Hershey and Martha Chase, 1953 Viruses can infect the E. coli bacteria. A virus has protein “head” and DNA core. Infection occurs when virus injects one of them into a bacterial cell. Which one is it?

9-7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Viruses infect bacteria Viral protein coat radioactively labeled (sulfur) Viral DNA radioactively labeled (phosphorus) Blended and centrifuged to Separate cells from virus Viral protein coats (radioactive) Bacteria with viral DNA (nonradioactive) Viral protein coats (nonradioactive) Bacteria with viral DNA (radioactive) Hershey-Chase experiment demonstrating DNA is the genetic material

9-8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA (deoxyribonucleic acid) is a chain of nucleotides Base - one of four types: adenine (A), thymine (T) guanine (G), cytosine (C) Sugar - deoxyribose Phosphate Nucleotides are composed of:

9-9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA Properties: Chargaff’s Findings Amount of A’s = Amount of T’s Amount of G’s = Amount of C’s. Other: Phosphate and sugar is hydrophilic Nitrogenous bases are hydrophobic

9-10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Rosalind Franklin’s X-Ray Crystallography data = Double Helix DNA Properties:

9-11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA Structure - Watson and Crick Built models

9-12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA bases pair via hydrogen bonds Validated Erwin Chargaff observations: # of adenine = # of thymine # of guanine = # of cytosine Complementary bases pair: A and T pair C and G pair

9-13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA exists as a double helix X-ray diffraction indicated DNA has a repeating structure and a regular width. -Maurice Wilkins and Rosalind Franklin DNA is double-stranded molecules wound in a double helix. -James Watson and Francis Crick C T G A C T G A C T G A C A G C T G A C T G

9-14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Orientation of DNA The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone. The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.

9-15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA is a double helix P A P C P G P T P C P G P A P C P T G P P C P A sugar and phosphate “backbone” connects nucleotides in a chain. P G P Two nucleotide chains together wind into a helix. DNA strands are antiparallel. DNA has directionality. 5’5’ 3’3’ 3’3’ 5’5’ Hydrogen bonds between paired bases hold the two DNA strands together.

9-16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Roles of the genetic material “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick

9-17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication the process of making new copies of the DNA molecules Test: Meselson and Stahl grew E. coli in media with heavy nitrogen and then in media with lighter nitrogen. Nitrogen becomes part of the DNA molecule as replication occurs. Potential mechanisms: organization of DNA strands Conservative old/old + new/new Semiconservative old/new + new/old Dispersive mixed old and new on each strand

9-18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Meselson and Stahl’s replication experiment Conclusion: Replication is semiconservative.

9-19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication as a process Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

9-20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chromatin = DNA and associated proteins DNA winds around histone proteins (nucleosomes). Other proteins wind DNA into more tightly packed form, the chromosome. Unwinding portions of the chromosome is important for mitosis, replication and making RNA.

9-21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Binding proteins prevent single strands from rewinding. Replication Helicase protein binds to DNA sequences called origins and unwinds DNA strands. 5’5’ 3’ 5’ 3’ Primase protein makes a short segment of RNA complementary to the DNA, a primer. 3’ 5’ 3’

9-22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication Overall direction of replication 5’ 3’ 5’5’ 5’ 3’ 5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

9-23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. 5’ Overall direction of replication 5’ 3’ 5’5’ DNA polymerase proofreads bases added and replaces incorrect nucleotides.

9-24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 5’ 3’ 5’5’ 5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction.

9-25 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 3’ 5’ 3’ 5’5’ 5’ 3’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

9-26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5’ Replication 5’ 3’ 5’5’ 5’ 3’ Overall direction of replication 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Okazaki fragment

9-27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

9-28 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 3’ 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

9-29 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 5’ 3’ 5’ 3’ 5’ 3’ 5’5’ 5’ Exonuclease enzymes remove RNA primers.

9-30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication Exonuclease enzymes remove RNA primers. Ligase forms bonds between sugar-phosphate backbone. 3’ 5’ 3’ 5’ 3’ 5’5’ 5’

9-31 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication 5’ 3’ 5’5’ 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ 5’5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’5’ 5’

9-32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Enzymes in DNA replication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase binds nucleotides to form new strands Ligase joins Okazaki fragments and seals other nicks in sugar- phosphate backbone Primase adds short primer to template strand Exonuclease removes RNA primer and inserts the correct bases

9-33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) Is a method in which multiple repetitions of DNA replication are performed in a test tube. Mix in test tube: DNA template DNA to be amplified Primers one complementary to each strand Nucleotides A, G, C, and T DNA polymerase heat stable form from thermophilic bacteria

9-34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) DNA replication in a test tube DNA template is denatured with heat to separate strands. CTTGATCGC 3’3’ 5’5’ GATCAA G CG 3’3’ 5’5’

9-35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) DNA template is denatured with heat to separate strands. CTTGATCGC 3’3’ 5’5’ GATCAA G CG 3’3’ 5’5’

9-36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) CTTGATCGC 3’3’ 5’5’ GATCAA G CG 3’3’ 5’5’ Each DNA primer anneals, binding to its complementary sequence on the template DNA C T T GCG 5’5’ 5’5’ 3’3’ 3’3’

9-37 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. CTTGATCGC 3’3’ 5’5’ GATCAA G CG 3’3’ 5’5’ C T T GCG 5’5’ 5’5’ 3’3’ C G C G A T G A A C T A 3’3’

9-38 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR)

9-39 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A gene: molecular definition A gene is a segment of DNA which directs the formation of RNA which in turn directs formation of a protein. Information is conveyed by the sequence of the nucleotides. The protein (or functional RNA) creates the phenotype.

9-40 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polymerase Chain Reaction (PCR) Denaturation Each DNA primer anneals, binding to its complementary sequence on the template DNA DNA template is denatured with high heat to separate strands. Annealing Extension DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. Multiple rounds of denaturation-annealing-extension are performed to create many copies of the template DNA between the two primer sequences.