Overview Lifes Operating Instructions In 1953 James Watson

Overview: Life’s Operating Instructions • In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA • DNA, the substance of inheritance, is the most celebrated molecule of our time • Hereditary information is encoded in DNA and reproduced in all cells of the body • This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Concept 16. 1: DNA is the genetic material • Early in the 20 th century, the identification of the molecules of inheritance loomed as a major challenge to biologists Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

The Search for the Genetic Material: Scientific Inquiry • When Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material • The key factor in determining the genetic material was choosing appropriate experimental organisms • The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Evidence That DNA Can Transform Bacteria • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 • Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a harmless “R” strain • When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic • He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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 RESULTS Mouse dies Mouse healthy Mouse dies Living S cells are found in blood sample

• In 1944, Oswald Avery, Maclyn Mc. Carty, and Colin Mac. Leod 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 • Many biologists remained skeptical, mainly because little was known about DNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Evidence That Viral DNA Can Program Cells • More evidence for DNA as the genetic material came from studies of a virus that infects bacteria • Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research Animation: Phage T 2 Reproductive Cycle Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T 2 • To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T 2 (DNA or protein) enters an E. coli cell during infection • They concluded that the injected DNA of the phage provides the genetic information Animation: Hershey-Chase Experiment Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -4 Phage Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch 1: Sulfur (35 S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: Phosphorus (32 P) Centrifuge Pellet Radioactivity (phage DNA) in pellet

Additional Evidence That DNA Is the Genetic Material • In 1947, Erwin Chargaff reported that DNA composition varies from one species to the next • This evidence of diversity made DNA a more credible candidate for the genetic material • By the 1950 s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group Animation: DNA and RNA Structure Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Building a Structural Model of DNA: Scientific Inquiry • After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role • Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure • Franklin produced a picture of the DNA molecule using this technique Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -6 Rosalind Franklin’s X-ray diffraction photograph 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 © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -7 5 end Hydrogen bond 3 end 1 nm 3. 4 nm 3 end 0. 34 nm Key features of DNA structure 5 end Partial chemical structure Space-filling model

• Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA • Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior • At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width • Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -UN 298 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

• Watson and Crick reasoned that the pairing was more specific, dictated by the base structures • They determined that adenine paired only with thymine, and guanine paired only with cytosine Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -8 Sugar Adenine (A) Sugar Thymine (T) Sugar Guanine (G) Cytosine (C)

Concept 16. 2: Many proteins work together in DNA replication and repair • The relationship between structure and function is manifest in the double helix • Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

The Basic Principle: 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 © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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.

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.

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.

LE 16 -9_4 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. The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand one new strand.

• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand • Competing models were the conservative model and the dispersive model Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

• Experiments by Meselson and Stahl supported the semiconservative model • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope • The first replication produced a band of hybrid DNA, eliminating the conservative model • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -11 Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) First replication Conservative model Semiconservative model Dispersive model Less dense More dense Second replication

DNA Replication: A Closer Look • The copying of DNA is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins participate in DNA replication Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Getting Started: Origins of Replication • Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” • 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 • At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -12 Parental (template) strand Origin of replication Bubble Daughter (new) strand 0. 25 µm Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

Animation: Origins of Replication Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Elongating a New DNA Strand • Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork • Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate • The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Antiparallel Elongation • The antiparallel structure of the double helix (two strands oriented 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork • 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Priming DNA Synthesis • DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end • The initial nucleotide strand is a short one called an RNA or DNA primer • An enzyme called primase can start an RNA chain from scratch • Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

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

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

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

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

LE 16 -15_6 Primase joins RNA nucleotides into a primer. 3 5 5 3 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 5 3 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5 3 3 5 5 3 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. 3 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. 5 3 3 5 Overall direction of replication

Other Proteins That Assist DNA Replication • Helicase untwists the double helix and separates the template DNA strands at the replication fork • 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 © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

• Primase synthesizes an RNA primer at the 5 ends of the leading strand the Okazaki fragments • DNA pol III continuously synthesizes the leading strand elongates Okazaki fragments • DNA pol I removes primer from the 5 ends of the leading strand Okazaki fragments, replacing primer with DNA and adding to adjacent 3 ends • DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand also joins the lagging strand fragments Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

The DNA Replication Machine as a Stationary Complex • The proteins that participate in DNA replication form a large complex, a DNA replication “machine” • The DNA replication machine is probably stationary during the replication process • Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules Copyright © 2005 Pearson Education, Inc. publishing as 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 • In nucleotide excision repair, enzymes cut out and replace damaged stretches of DNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

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 © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -18 End of parental DNA strands 5 Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New leading 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16 -19 1 µm

• 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings