BIOL 2107 Chapter 13

DNA and Hereditary Information

• In 1953, Watson and Crick introduced a double-helical model for the structure of DNA

• Hereditary information in DNA directs the development of your biochemical, anatomical, physiological, and to some extent behavioral traits

• Hereditary information is reproduced in all cells of the body during DNA replication

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

• Later work by Oswald Avery and others identified the transforming substance as 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

• A virus is DNA (or RNA) enclosed by a protective coat, usually made of protein

• Viruses must infect cells and take over the cells’ metabolic machinery in order to reproduce

• In 1952, Alfred Hershey and Martha Chase showed that DNA is the genetic material of a phage Known as T2

• They designed an experiment showing that only the DNA of the T2 phage, and not the 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

• In 1950, 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

• Two findings became known as Chargaff’s rules

  • The base composition of DNA varies between species

  • In any species the percentages of A and T bases are equal and the percentages 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

• James Watson and Francis Crick were first to determine the structure of DNA

• 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-ray measurements and the 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)

• 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 = the amount of T, and the amount of G = the amount of C

The Basic Principle: Base Pairing to a Template Strand

• Since the two strands of DNA are complementary, each strand stores the information necessary to reconstruct the other

• 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 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)

Getting Started with DNA Replication

• Replication begins at sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”

• At each end of a bubble is a replication fork, a Y-shaped region where the parental strands of DNA are being unwound

• For the long DNA molecules in eukaryotes, multiple replication bubbles form and eventually fuse, speeding up the copying of DNA

• Several kinds of proteins participate in the unwinding

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

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

  • Topoisomerase relieves the strain caused by tight twisting ahead of the replication fork by breaking, swiveling, and rejoining DNA strands

Synthesizing a New DNA Strand

• Enzymes that synthesize DNA cannot initiate synthesis of polynucleotide; they can only add nucleotides to an already existing chain bas-paired with the template

• The initial nucleotide strand is a short RNA primer

• The enzyme, primase, starts an RNA chain with a single RNA nucleotide and adds RNA nucleotides one at a time using the parental DNA as a template

• The primer is a short (5-10 nucleotides long)

• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork

• They add nucleotides to the 3’ end of a preexisting chain

• 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 consists of a sugar attached to a base and to three phosphate groups

• dATP is used to make DNA and is similar to the ATP of energy metabolism

• The difference is in the sugars: dATP has deoxyribose, whereas ATP has ribose

• As each monomer nucleotide joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate

Antiparallel Elongation

• Newly replicated DNA strands must be formed antiparallel to the template strand

• Because DNA polymerases add nucleotides only to the free 3’ end of a growing strand, the 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

• Only one primer is required to synthesize the leading strand

• To elongate the other new strand, 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

• These are 100-200 nucleotides long in eukaryotes and 1,000-2,000 nucleotides long in E. coli

• After formation of Okazaki fragments, DNA polymerase I removes the RNA primers and replaces the nucleotides with DNA

• The remaining gaps are joined together by DNA ligase

Proofreading and Repairing DNA

• Errors in the completed DNA molecule amount to only one in 10 billion

• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides

• In mismatch repair of DNA, other enzymes correct errors in base pairing

• A hereditary defect in one such enzyme is associated with a form of colon cancer

• This defect allows cancer-causing errors to accumulate in DNA faster than normal

• DNA can be damaged by exposure to harmful chemical or physical agents, such as X-rays

• DNA bases can also undergo spontaneous changes

• In many cases a nuclease cuts out and replaces damaged stretches of DNA

• One such DNA repair system is called nucleotide excision repair

• DNA repair enzymes in our skin repair genetic damage caused by the UV light of sunlight

Evolutionary Significance of Altered DNA Nucleotides

• The error rate after proofreading repair is extremely 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

• For linear DNA, the usual replication machinery cannot complete the 5’ ends of daughter strands

• Repeated rounds of replication produce shorter DNA molecules with uneven ends

• Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres

• Telomeres do not contain genes; they typically consist of multiple repetitions of one short nucleotide sequence

• Telomeres do not prevent the shortening of DNA molecules, but they do postpone it

• 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

• Telomerase is not active in most human somatic cells, however, it does show inappropriate activity in some cancer cells

• Telomerase is currently under study as a target for cancer therapies

A Chromosome Consists of a DNA Molecule Packed Together with Proteins

• 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 striking changes in the degree of packing during the course of the cell cycle

• Proteins called histones are responsible for the first level of DNA packing in chromatin

• Four types of histones are most common in chromatin

• A nucleosome consists of DNA wound twice around a protein core of eight histones, wo of each pf the main histone types

• Interphase chromatin is generally much less condensed but the 10-nm fiber may be further compacted and also folded into looped domains

• Even during interphase, centrosomes and other parts of chromosomes are highly condensed, similar to metaphase chromosomes

• This condensed chromatin is called heterochromatin; the dispersed, less compacted chromatin is called euchromatin

• Dense packing of the heterochromatin makes it largely inaccessible to the machinery responsible for transcribing genetic information

• Chromosomes are dynamic in structure; a condensed region may be condensed loosened, modified, and remodeled as needed for various cell processes

• Histones can undergo chemical modifications that result in changes in chromatin organization

DNA Cloning: Making Multiple Copies of a Gene or other DNA Segment

• To work directly with specific genes, scientists prepare well-defined segments of DNA in identical copies, a process called DNA cloning

• Most methods of cloning pieces of DNA in the laboratory share general features

• Many bacteria contain plasmids, small circular DNA molecules that replicate separately from the bacterial chromosome

• To clone pieces of DNA using bacteria, researchers first obtain a plasmid and insert DNA from another source (“foreign DNA”) into it

• The resulting plasmid is called recombinant DNA

Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in Cloning

• The PCR can produce many copies of a specific target segment of DNA

• A three-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

• The key to PCR is an unusual, heat stable DNA polymerase called Taq polymerase

• During each PCR cycle the reaction mixture is heated to separate the DNA strands

• Then it is cooled to allow annealing of short, single-stranded DNA primers complementary to sequences at the ends of the target segment

• A DNA polymerase extends the primers in the 5’ to 3’ direction

• PCR amplification alone cannot substitute for gene cloning in cells

• Instead, PCR is used to provide the specific DNA fragment to be cloned

• PCR primers are synthesized to include a restriction site that matches the site in the cloning vector

• The fragment and vector are cut and ligated together

• PCR has had a major impact on biological research and genetic engineering

• It has been used to amplify DNA from a wide variety of sources

DNA Sequencing

• Once a gene is cloned, complementary base pairing can be exploited to determine the gene’s complete nucleotide sequence; this is called DNA sequencing

• “Next-generation” sequencing techniques, developed in the few decades, are rapid and inexpensive

• They sequence by synthesizing the complementary strand of a single, immobilized template strand

• A chemical technique enables electronic monitors to identify which nucleotide is being added at each step