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

 

Concept 16.1: DNA is the genetic material

            Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists

 

The Search for the Genetic Material: Scientific Inquiry

            When T. H. 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, one pathogenic and one harmless

            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

            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 viruses that infect bacteria

            Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research

            In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2

            To determine this, 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

Additional Evidence That DNA Is the Genetic Material

            Two findings became known as Chargaff’s rules

         The base composition of DNA varies between species

         In any species the number of A and T bases are equal and the number of G and C bases are equal

            The basis for these rules was not understood until the discovery of the double helix

 

Building a Structural Model of DNA: Scientific Inquiry

            After DNA was accepted as the genetic material, the challenge was to determine how its structure accounts for its role in heredity

            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

            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 pattern in the photo 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 outer  sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

            Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions)

            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 data

            Watson and Crick reasoned that the pairing was more specific, dictated by the base structures

            They 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

 

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

 

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

            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 (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)

            Experiments by Matthew Meselson and Franklin 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

 

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

            Replication begins at particular 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

            Helicases are enzymes that untwist the double helix at the replication forks

            Single-strand binding proteins bind to and stabilize single-stranded DNA

            Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

            DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3Ę end

            The initial nucleotide strand is a short RNA primer

            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 3Ę end serves as the starting point for the new DNA strand

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

            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

Antiparallel Elongation

            The antiparallel structure of the double helix affects replication

            DNA polymerases add nucleotides only to the free 3Ę end of a growing strand; therefore, a new DNA strand can elongate only in the 5Ę to 3Ę direction

            Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork

 

The DNA Replication Complex

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 exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes

            In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA

 

Evolutionary Significance of Altered DNA Nucleotides

            Error rate after proofreading repair is low but not zero

            Sequence changes may become permanent and can be passed on to the next generation

            These changes (mutations) are the source of the genetic variation upon which natural selection operates

 

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 5Ę ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends

            This is not a problem for prokaryotes, most of which have circular chromosomes

            Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends 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

 

Concept 16.3 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, a complex of DNA and protein, is found in the nucleus of eukaryotic cells

            Chromosomes fit into the nucleus through an elaborate, multilevel system of packing

            Chromatin undergoes changes in packing during the cell cycle

            At interphase, some chromatin is organized into a 10-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping

            Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus

            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