The Molecular Basis of Inheritance Chapter 16 The Molecular Basis of Inheritance

Describe the structure of DNA Learning Targets: Describe the structure of DNA Describe the process of DNA replication; include the following terms: antiparallel structure, DNA polymerase, leading strand, lagging strand, Okazaki fragments, DNA ligase, primer, primase, helicase, topoisomerase, single-strand binding proteins Describe the function of telomeres Compare a bacterial chromosome and a eukaryotic chromosome Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Phenomenon termed transformation DNA a timeline The genetic role of DNA began with research by Frederick Griffith in 1928 He worked with two strains of a bacterium, one pathogenic and one harmless When heat-killed remains of the pathogenic strain were mixed with living cells of the harmless strain some cells became pathogenic Phenomenon termed transformation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Griffith EXPERIMENT RESULTS Mixture of heat-killed S cells and living R cells Griffith EXPERIMENT Living S cells (control) Living R cells (control) Heat-killed S cells (control) RESULTS Figure 16.2 Can a genetic trait be transferred between different bacterial strains? Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells

DNA a timeline continued In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Evidence That Viral DNA Can Program Cells More evidence for DNA as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research Animation: Phage T2 Reproductive Cycle Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Evidence That DNA Is the Genetic Material In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 They concluded that the injected DNA of the phage provides the genetic information Animation: Hershery-Chase Experiment Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Chase and Hershey EXPERIMENT Empty protein shell Radioactivity (phage protein) in liquid Radioactive protein Phage Bacterial cell Batch 1: radioactive sulfur (35S) DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Figure 16.4 Is protein or DNA the genetic material of phage T2? Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet

Evidence That DNA Is the Genetic Material It was known that DNA is a polymer of nucleotides( Sugar, Phosphate, Base) In 1950, Erwin Chargaff showed DNA composition varies from species to species Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases Animation: DNA and RNA Structure Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Sugar–phosphate backbone 5 end Sugar (deoxyribose) 3 end Nitrogenous bases Thymine (T) Adenine (A) Figure 16.5 The structure of a DNA strand Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose) 3 end Guanine (G)

Building a Structural Model of DNA After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role Rosalind Franklin produced a picture of the DNA molecule using a technique called X-ray crystallography Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Building a Structural Model of DNA Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases The width suggested that the DNA molecule was made up of two strands, forming a double helix Animation: DNA Double Helix Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Figure 16.7 The double helix 3 end 0.34 nm 5 end

Watson and Crick’s contribution determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Figure 16.8 Base pairing in DNA Guanine (G) Cytosine (C)

Base Pairing to a Template Strand Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules Animation: DNA Replication Overview Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Base Pairing to a Template Strand C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C Parent molecule Separation of strands “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept

Animation: Origins of Replication DNA Replication Replication begins at special sites called origins of replication More than a dozen enzymes and other proteins participate in DNA replication A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied Animation: Origins of Replication Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Origins of replication in E. coli Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double-stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules Figure 16.12 Origins of replication in E. coli and eukaryotes

Origins of replication in eukaryotes Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Bubble Replication fork Figure 16.12 Origins of replication in E. coli and eukaryotes Two daughter DNA molecules

DNA Replication At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

The initial nucleotide strand is a short RNA primer DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure 16.13 Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase The initial nucleotide strand is a short RNA primer

DNA Replication An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Synthesizing a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Most DNA polymerases require a primer and a DNA template strand The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Synthesizing a New DNA Strand Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate dATP supplies adenine to DNA and is similar to the ATP of energy metabolism The difference is in their sugars: dATP has deoxyribose while ATP has ribose As each monomer of dATP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nucleoside triphosphate New strand 5 end Template strand 3 end 5 end 3 end Sugar A T A T Base Phosphate C G C G G C G C DNA polymerase 3 end A T A Figure 16.14 Incorporation of a nucleotide into a DNA strand T 3 end C Pyrophosphate C Nucleoside triphosphate 5 end 5 end

Antiparallel Elongation The antiparallel structure of the double helix (two strands in opposite directions) affects replication DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3direction Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork Animation: Leading Strand Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Overall directions of replication Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 DNA poll III Parental DNA Figure 16.15 Synthesis of the leading strand during DNA replication 3 5 5 3 5

Animation: Lagging Strand To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase Animation: Lagging Strand Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Overall directions of replication Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Leading strand Figure 16.6 Synthesis of the lagging strand Overall directions of replication

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand Figure 16.6 Synthesis of the lagging strand

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 Figure 16.6 Synthesis of the lagging strand

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 1 5 Figure 16.6 Synthesis of the lagging strand

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 Figure 16.6 Synthesis of the lagging strand

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 5 3 Figure 16.6 Synthesis of the lagging strand 3 5 2 1

Figure 16.6 Synthesis of the lagging strand 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 5 3 Figure 16.6 Synthesis of the lagging strand 3 5 2 1 5 3 3 1 5 2 Overall direction of replication

Single-strand binding protein Overall directions of replication Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Helicase Leading strand 5 DNA pol III 3 3 Primer Primase 5 Parental DNA 3 Figure 16.17 A summary of bacterial DNA replication DNA pol III Lagging strand 5 DNA pol I DNA ligase 4 3 5 3 2 1 3 5

The DNA Replication Complex The proteins that participate in DNA replication form a large complex, a “DNA replication machine” Animation: DNA Replication Review Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nuclease DNA polymerase DNA ligase Figure 16.18 Nucleotide excision repair of DNA damage DNA ligase

Replicating the Ends of DNA Molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Figure 16.19 Shortening of the ends of linear DNA molecules 5 Ends of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication Figure 16.19 Shortening of the ends of linear DNA molecules 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules

Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules It has been proposed that the shortening of telomeres is connected to aging Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Chromosome makeup The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Animation: DNA Packing Chromosome makeup Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Histones are proteins that are responsible for the first level of DNA packing in chromatin For the Cell Biology Video Cartoon and Stick Model of a Nucleosomal Particle, go to Animation and Video Files. Animation: DNA Packing Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Chromosome makeup DNA, the double helix Histones Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histone tail Histones Figure 16.21a Chromatin packing in a eukaryotic chromosome DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Chromosome makeup 30-nm fiber Looped domains (300-nm fiber) Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber Figure 16.21b Chromatin packing in a eukaryotic chromosome Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome

Loosely packed chromatin is called euchromatin Chromosome makeup Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Chromosome modifications Histones can undergo chemical modifications that result in changes in chromatin organization For example, phosphorylation of a specific amino acid on a histone tail affects chromosomal behavior during meiosis Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings