Overview: Life’s Operating Instructions

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

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

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

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

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 Many biologists remained skeptical, mainly because little was known about DNA

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

Phage head Tail Tail fiber DNA 100 nm Bacterial cell

In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information

Empty protein shell Radioactive protein Radioactivity (phage protein) in liquid Phage Bacterial cell Batch 1: Sulfur (35S) DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Batch 2: Phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet 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 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group

Nucleotide monomers are made up of nucleosides and phosphate groups
Nucleoside = nitrogenous base + sugar There are two families of nitrogenous bases: Pyrimidines have a single six-membered ring Purines have a six-membered ring fused to a five-membered ring In DNA, the sugar is deoxyribose In RNA, the sugar is ribose

Nitrogenous bases Pyrimidines Purines Pentose sugars Cytosine C Thymine (in DNA) T Uracil (in RNA) U Adenine A Guanine G Deoxyribose (in DNA) Ribose (in RNA)

Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose) 3 end 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

Rosalind Franklin 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

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

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

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

Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C)

5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Key features of DNA structure Partial chemical structure Space-filling model

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

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

Nucleotide Polymers Nucleotide polymers are linked together, building a polynucleotide Adjacent nucleotides are joined by covalent bonds that form between the –OH group on the 3´ carbon of one nucleotide and the phosphate on the 5´ carbon on the next These links create a backbone of sugar-phosphate units with nitrogenous bases as appendages The sequence of bases along a DNA or mRNA polymer is unique for each gene

The DNA Double Helix A DNA molecule has two polynucleotides spiraling around an imaginary axis, forming a double helix In the DNA double helix, the two backbones run in opposite 5´ to 3´ directions from each other, an arrangement referred to as antiparallel One DNA molecule includes many genes The nitrogenous bases in DNA form hydrogen bonds in a complementary fashion: A always with T, and G always with C

Sugar-phosphate backbone 3¢ end 5¢ end Base pair (joined by hydrogen bonding) Old strands Nucleotide about to be added to a new strand New strands

DNA and Proteins as Tape Measres of Evolution
The linear sequences of nucleotides in DNA molecules are passed from parents to offspring Two closely related species are more similar in DNA than are more distantly related species Molecular biology can be used to assess evolutionary kinship

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 and 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

First replication Second replication Parent cell 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.

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

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

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

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

Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double- stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand Figure Origins of replication in E. coli and eukaryotes 0.25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

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

New strand Template strand 5¢ end 3¢ end 5¢ end 3¢ end Sugar Base Phosphate DNA polymerase 3¢ end 3¢ end Pyrophosphate Nucleoside triphosphate 5¢ 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

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

3¢ Parental DNA 5¢ 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

Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 3¢ RNA primer 3¢ 5¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 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. 5¢ 3¢ 3¢ 5¢ DNA pol I replaces the RNA with DNA, adding to the 3¢ end of fragment 2. 5¢ 3¢ 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

Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments
DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments DNA pol I removes primer from the 5 ends of the leading strand and 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 and also joins the lagging strand fragments

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

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

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

LE 16-17 A thymine dimer distorts the DNA molecule.
ligase 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. LE 16-17

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

LE 16-18 End of parental DNA strands 5¢ 3¢ Lagging strand
Last fragment RNA primer Leading strand Previous fragment 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 Second round of replication Further rounds New leading strand Shorter and shorter daughter molecules LE 16-18 5¢ 3¢ 5¢

LE 16-19 1 µm

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

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

A chromosome consists of a DNA molecule packed together with proteins
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

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.

Nucleosomes, or “beads on a string” (10-nm fiber)
(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)

Looped domains (300-nm fiber) Metaphase chromosome
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

Chromatin is organized into fibers 10-nm fiber
DNA winds around histones to form nucleosome “beads” Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber

300-nm fiber Metaphase chromosome
The 30-nm fiber forms looped domains that attach to proteins Metaphase chromosome The looped domains coil further The width of a chromatid is 700 nm

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

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

Animations and Videos Bozeman - What is DNA? Griffith's Experiment Hershey-Chase Experiment Meselson-Stahl Experiment – 1 Meselson-Stahl Experiment – 2 Bozeman - Meselson and Stahl Experiment Structural Organization of the Chromosome Replication of a Chromosome DNA Structure and Replication Structural Basis of DNA Replication

Animations and Videos DNA Replication Fork How Nucleotides Are Added In DNA Replication DNA Replication Bidirectional Replication of DNA Proofreading Function of DNA Bozeman - DNA Replication Addition and Deletion Mutations Mutation by Base Substitution Slipped-strand Mispairing Thymine Dimers

Animations and Videos Screening Mutations Bozeman – Mutations Genetic Testing - Sickle-Cell Anemia Transposons: Shifting Segments of the Genome Mechanism of Transposition Telomerase – 1 Telomerase – 2 Chapter Quiz Questions – 1 Chapter Quiz Questions – 2

Who conducted the X-ray diffraction studies that were key to the discovery of the structure of DNA?
Griffith Franklin Meselson and Stahl Chargaff McClintock Answer: B

Who conducted the X-ray diffraction studies that were key to the discovery of the structure of DNA?
Griffith Franklin Meselson and Stahl Chargaff McClintock 69

How do the leading and the lagging strands differ?
The leading strand is synthesized in the same direction as the movement of the replication fork, whereas the lagging strand is synthesized in the opposite direction. The leading strand is synthesized at twice the rate of the lagging strand. The lagging strand is synthesized continuously, whereas the leading strand is synthesized in short fragments that are ultimately stitched together. The leading strand is synthesized by adding nucleotides to the 3' end of the growing strand, whereas the lagging strand is synthesized by adding nucleotides to the 5' end. Answer: A DNA is made only in the 5' to 3' direction. 70

How do the leading and the lagging strands differ?
The leading strand is synthesized in the same direction as the movement of the replication fork, whereas the lagging strand is synthesized in the opposite direction. The leading strand is synthesized at twice the rate of the lagging strand. The lagging strand is synthesized continuously, whereas the leading strand is synthesized in short fragments that are ultimately stitched together. The leading strand is synthesized by adding nucleotides to the 3' end of the growing strand, whereas the lagging strand is synthesized by adding nucleotides to the 5' end. 71

What kind of evidence about the structure of DNA came from each of the following branches of science? physics chemistry biology Answer: X-ray crystallography, B. The nature of ribose sugar, purines, and pyrimidines, C. Data from Chargaff on the ratios between A and T and so on, 72

What kind of evidence about the structure of DNA came from each of the following branches of science? physics: X-ray crystallography chemistry: The nature of ribose sugar, purines, and pyrimidines biology: Data from Chargaff on the ratios between A and T and so on Answer: X-ray crystallography, B. The nature of ribose sugar, purines, and pyrimidines, C. Data from Chargaff on the ratios between A and T and so on, 73

If the result of the Hershey and Chase experiment had been that radioactive sulfur (35S) was found inside the cells instead of radioactive phosphorus (32P), what could have been concluded? Answer: It would have been concluded that protein functions as the genetic material (this, of course, did not occur). Figure 13.4. 74

If the result of the Hershey and Chase experiment had been that radioactive sulfur (35S) was found inside the cells instead of radioactive phosphorus (32P), what could have been concluded? It would have been concluded that protein functions as the genetic material (this, of course, did not occur). Answer: It would have been concluded that protein functions as the genetic material (this, of course, did not occur). Figure 13.4. 75

Define and diagram “semiconservative” as it applies to DNA replication.
Answer: A newly replicated DNA has one strand that had been present in the original and one completely new strand. Concept 13.2. 76

Telomeres, or the ends of linear chromosomes, have special structure and function, even though they are noncoding. Describe their structure and function. Answer: Figure 13.20, Concept Telomeres may have a major impact on students’ lives, as they may hold the secret to longer life. 77

What enzyme does a gamete-producing cell include that compensates for replication-associated shortening? DNA polymerase II ligase telomerase DNA nuclease proofreading enzyme Answer: C 78

What enzyme does a gamete-producing cell include that compensates for replication-associated shortening? DNA polymerase II ligase telomerase DNA nuclease proofreading enzyme 79

Which of the following is true of heterochromatin but not of euchromatin?
It is accessible to enzymes needed for gene expression. It becomes less tightly compacted after cell division. It includes DNA primarily found in expressed genes. It appears more pale when observed microscopically. It remains tightly coiled at the G1 phase. Answer: E 80

Which of the following is true of heterochromatin but not of euchromatin?
It is accessible to enzymes needed for gene expression. It becomes less tightly compacted after cell division. It includes DNA primarily found in expressed genes. It appears more pale when observed microscopically. It remains tightly coiled at the G1 phase. 81

Which of the following results from Griffith’s experiment is an example of transformation?
Mouse dies after being injected with living S cells. Mouse is healthy after being injected with living R cells. Mouse is healthy after being injected with heat-killed S cells. Mouse dies after being injected with a mixture of heat-killed S and living R cells. In blood samples from the mouse in D, living S cells were found. Answer: D

Which of the following results from Griffith’s experiment is an example of transformation?
Mouse dies after being injected with living S cells. Mouse is healthy after being injected with living R cells. Mouse is healthy after being injected with heat-killed S cells. Mouse dies after being injected with a mixture of heat-killed S and living R cells. In blood samples from the mouse in D, living S cells were found.

The X-ray data suggested that the double helix had a uniform diameter.
Nitrogenous bases are paired in specific combinations. Which of the following does not provide evidence to support this conclusion? A purine-purine pair is too wide to account for the 2-nm diameter of the double helix. A pyrimidine-pyrimidine pair is too narrow to account for the 2-nm diameter of the double helix. The X-ray data suggested that the double helix had a uniform diameter. Whenever one strand of DNA has an A, the partner strand has a T. The pairs of nitrogenous bases are held together by hydrogen bonds. Answer: E

Nitrogenous bases are paired in specific combinations
Nitrogenous bases are paired in specific combinations. Which of the following does not provide evidence to support this conclusion? A purine-purine pair is too wide to account for the 2-nm diameter of the double helix. A pyrimidine-pyrimidine pair is too narrow to account for the 2-nm diameter of the double helix. The X-ray data suggested that the double helix had a uniform diameter. Whenever one strand of DNA has an A, the partner strand has a T. The pairs of nitrogenous bases are held together by hydrogen bonds.

Scientific Skills Exercise
Tables like the one shown here are useful for organizing sets of data representing a common set of values (in this case, percentages of A, G, C, and T) for a number of different samples (in this case, species).

Does the distribution of bases in sea urchin DNA and salmon DNA follow Chargaff’s rules?
Yes, because the %A + %T is greater than the %G + %C in both species. No, because %A + %T does not equal %G + %C in both species. Yes, because the %A approximately equals the %T and the %G approximately equals the %C in both species. No, because %A is higher than %T and %G is higher than %C in both species. Answer: C

What is the %T in wheat DNA?
approximately 22% approximately 23% approximately 28% approximately 45% Answer: C

What is the %T in wheat DNA?
approximately 22% approximately 23% approximately 28% approximately 45%

Overview: Life’s Operating Instructions

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