9 DNA and Its Role in Heredity Chapter 9 DNA and Its Role in Heredity Key Concepts 9.1 DNA Structure Reflects Its Role as the Genetic Material 9.2 DNA Replicates Semiconservatively 9.3 Mutations Are Heritable Changes in DNA Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material By the early 20th century, a “chromosomal theory of inheritance” had been developed, proposing that Mendel’s genes are on the chromosomes. Then evidence began to accumulate indicating that DNA is the genetic material. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Circumstantial evidence: • DNA is present in the cell nucleus and in chromosomes. • It doubles during S phase of the cell cycle. • There is twice as much in diploid cells as in haploid cells. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material DNA was first isolated in 1868 from white blood cell nuclei. The young Swiss researcher called the fibrous substance “nuclein,” and proposed that it was the genetic material. It was composed of C, H, O, N, and P. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Dyes were developed in the early 20th century that showed color when bound to DNA in dividing cells. Figure 9.1 DNA in the Cell Cycle Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Chromosomes contain DNA, but also proteins, so scientists had to rule out proteins as the genetic material. In transformation experiments, it was shown that DNA from one strain of bacterium could genetically transform another strain: strain A + strain B DNA → bacterium strain B Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Viruses such as bacteriophage contain DNA and a little protein. Experiments showed that when a virus infects a bacterium, it injects only its DNA. Since the viral DNA genetically transforms the bacteria, this was further evidence for DNA as the genetic material. Figure 9.2 Viral DNA and Not Protein Enters Host Cells Figure 9.3 Transformation of Eukaryotic Cells (Part 1) Figure 9.3 Transformation of Eukaryotic Cells (Part 2) Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Egg cells can also be transformed in this way, resulting in a whole new genetically transformed organism—called transgenic. These methods form the basis of much applied research, including biotechnology and genetic engineering, and have provided strong evidence for DNA as the genetic material. Figure 9.4 X-Ray Crystallography Helped Reveal the Structure of DNA Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Chemical composition: Biochemists knew that nucleotides consisted of the sugar deoxyribose, a phosphate group, and nitrogen-containing bases: • Purines: adenine (A) and guanine (G) • Pyrimidines: cytosine (C) and thymine (T) • (Pneumonic: CARS get parked in the GARAGE, APPLES grow on TREES) Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material In 1950, Erwin Chargaff found the amount of A always equaled the amount of T, and amount of G always equaled the amount of C. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material 3-D model building: Francis Crick and James Watson combined all the knowledge of DNA to determine its structure: Double Helix Franklin’s X-ray crystallography convinced them the molecule was helical. Density measurements suggested there are two polynucleotide chains in the molecule. Modeling showed that DNA strands must be antiparallel. Figure 9.5 DNA Is a Double Helix (Part 1) Figure 9.5 DNA Is a Double Helix (Part 2) Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Watson and Crick suggested that: • The bases (B) are on the interior of the two strands, with a sugar-phosphate backbone on the outside. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material • Per Chargaff’s rule, a purine on one strand is paired with a pyrimidine on the other, making the base pairs (A–T and G–C) the same width down the helix. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material Four key features of DNA structure: • Double-stranded helix of uniform diameter The chains are held together by hydrogen bonds between the base pairs and by van der Waals forces between adjacent bases on the same strand. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material • The two strands are antiparallel. In the sugar–phosphate backbone, the phosphate groups are bonded to the 5ʹ carbon of one sugar and the 3ʹ carbon of the next. Figure 3.4 DNA Figure 9.5 DNA Is a Double Helix (Part 2) Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material The surfaces of A–T and G–C base pairs are chemically distinct. Binding of proteins to specific base pair sequences is key to DNA– protein interactions, which are necessary for replication and gene expression. Concept 9.1 DNA Structure Reflects Its Role as the Genetic Material DNA structure is essential to its functions: • Storage of genetic information • Precise replication during cell division by complementary base pairing • Susceptibility to mutations (stable changes in the genetic material) • Expression of the coded information as the phenotype Concept 9.2 DNA Replicates Semiconservatively Semiconservative replication: each parental strand is a template for a new strand. Concept 9.2 DNA Replicates Semiconservatively Two general steps in DNA replication: • The double helix is unwound, making two template strands available for new base pairing. • Nucleotides form base pairs with template strands and are linked together by phosphodiester bonds. Figure 9.7 Each New DNA Strand Grows by the Addition of Nucleotides to Its 3ʹ End Concept 9.2 DNA Replicates Semiconservatively DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs) or deoxyribonucleotides. During synthesis, two of the phosphate groups are released, and the final nucleotide is a monophosphate (adenine, thymine, cytosine, or guanine). Release of the two outer phosphate groups provides energy for formation of a phosphodiester bond. Concept 9.2 DNA Replicates Semiconservatively DNA replication begins with the binding of a large protein complex (pre-replication complex) to a specific site on the DNA molecule called the origin of replication (ori). The complex contains DNA polymerase, which catalyzes addition of nucleotides. Concept 9.2 DNA Replicates Semiconservatively In prokaryotes, the pre-replication complex binds to ori, the DNA unwinds, and replication proceeds in two directions. The replication fork is the site where DNA unwinds. Opening of each fork is catalyzed by DNA helicase. Figure 9.8 The Origin of DNA Replication Concept 9.2 DNA Replicates Semiconservatively Eukaryotic chromosomes are much longer, are linear, and have multiple origins of replication, which speed up replication. It would take weeks to fully replicate a chromosome from a single ori. Concept 9.2 DNA Replicates Semiconservatively DNA replication begins with a short primer or starter strand, usually a short single strand of RNA. The primer is complementary to the DNA template and is synthesized by a primase. DNA polymerase then adds nucleotides to the 3ʹ end of the primer and continues until replication of that section is completed. Figure 9.9 DNA Forms with a Primer Concept 9.2 DNA Replicates Semiconservatively DNA polymerases are very large and shaped like an open right hand. The “palm” brings the active site and the substrates into contact. The “fingers” recognize the nucleotide bases. The enzyme then changes shape and catalyzes formation of a new phosphodiester bond. Figure 9.10 DNA Polymerase Binds to the Template Strand Concept 9.2 DNA Replicates Semiconservatively The leading strand is oriented to grow continuously at its 3′ end as the fork opens. The lagging strand is oriented so that its exposed 3′ end gets farther from the fork. Synthesis of the lagging strand occurs in small, discontinuous stretches called Okazaki fragments. Figure 9.11 The Two New Strands Form in Different Ways Concept 9.2 DNA Replicates Semiconservatively Each Okazaki fragment requires its own primer. DNA polymerase adds nucleotides to the 3′ end until reaching the primer of the previous fragment. A different DNA polymerase then replaces the primer with DNA. The final phosphodiester linkage between fragments is catalyzed by DNA ligase. Figure 9.12 The Lagging Strand Story (Part 1) Figure 9.12 The Lagging Strand Story (Part 2) Concept 9.2 DNA Replicates Semiconservatively DNA polymerase works very fast but makes very few errors. It is processive—it catalyzes many sequential polymerization reactions each time it binds to DNA. A DNA polymerase can add thousands of nucleotides before it detaches from DNA. Figure 9.13 Telomeres and Telomerase Concept 9.2 DNA Replicates Semiconservatively Chromosome ends must be protected from being joined to other chromosomes by the DNA repair system. Telomeres are repetitive sequences at the ends of eukaryotic chromosomes. The repeats bind a protein complex called shelterin, which protects the ends from being joined together. The repeats also form loops, which are also protective. Figure 9.13 Telomeres and Telomerase Concept 9.2 DNA Replicates Semiconservatively After 20–30 cell divisions, the chromosome ends become too short, the chromosomes lose their integrity, and apoptosis ensues. But continuously dividing cells like bone marrow and gametes maintain their telomeric DNA. • Telomerase catalyzes the addition of lost telomeric sequences. It has an RNA sequence that acts as a template for the telomeric DNA. Concept 9.2 DNA Replicates Semiconservatively Telomere lengths tend to shorten with aging. If a gene expressing high levels of telomerase is added to human cells in culture, their telomeres do not shorten, and the cells become immortal. This is also seen in mice that overexpress telomerase—they live longer. Cancer cells also express telomerase. Concept 9.2 DNA Replicates Semiconservatively DNA polymerases can make mistakes in replication, but most errors are repaired. Two major repair mechanisms: • Proofreading—DNA polymerase has a proofreading function, and if bases are paired incorrectly, the nucleotide is removed. • Mismatch repair—after replication, other proteins scan for mismatched bases missed in proofreading and replace them with correct ones. Figure 9.14 DNA Repair Mechanisms Concept 9.2 DNA Replicates Semiconservatively Copies of DNA sequences can be made by the polymerase chain reaction (PCR) using: • A double-stranded DNA sample • Two primers complementary to the ends of the sequence to be copied • The four dNTPs • A DNA polymerase that works at high temperatures • Salts and a buffer to maintain pH Figure 9.15 The Polymerase Chain Reaction Concept 9.3 Mutations Are Heritable Changes in DNA Mutations are changes in the nucleotide sequence of DNA that are passed on from one cell or organism to another. Mutations occur by a variety of processes, including replication errors that are not corrected by repair systems. Concept 9.3 Mutations Are Heritable Changes in DNA Somatic mutations occur in somatic (body) cells. They are passed on by mitosis but not to sexually produced offspring. Germ line mutations occur in germ line cells that give rise to gametes. A gamete with a mutation passes it on to the new organism at fertilization. Mutations may or may not affect the phenotype. Concept 9.3 Mutations Are Heritable Changes in DNA Silent mutations do not affect protein function. Loss of function mutations prevent gene transcription or produce nonfunctional proteins; nearly always recessive. Gain of function mutations lead to a protein with altered function. Usually dominant; common in cancer cells. Figure 9.16 Mutation and Phenotype Concept 9.3 Mutations Are Heritable Changes in DNA Conditional mutations cause phenotypes under restrictive conditions, such as temperature (e.g., point restriction coat color in cats and rabbits). The wild-type phenotype is expressed under other, permissive conditions. Concept 9.3 Mutations Are Heritable Changes in DNA A point mutation results from the gain, loss, or substitution of a single nucleotide. • Can arise from replication errors or be caused by environmental mutagens such as radiation or certain chemicals. Concept 9.3 Mutations Are Heritable Changes in DNA Point mutations may alter the amino acid sequence in a protein with drastic effects. The sickle-cell disease allele differs from the normal by one base pair, resulting in a polypeptide with only one different amino acid. Concept 9.3 Mutations Are Heritable Changes in DNA Chromosomal mutations are extensive changes in genetic material involving whole chromosomes. They can result from mutagens or drastic errors in replication. They can provide new combinations of genes and genetic diversity important to evolution by natural selection. Concept 9.3 Mutations Are Heritable Changes in DNA Chromosomal mutations: • Deletions—loss of a chromosome segment; can have severe or fatal consequences • Duplications—homologous chromosomes break in different places and recombine with wrong partners; one may have two copies of the segment and the other may have none Concept 9.3 Mutations Are Heritable Changes in DNA Inversions result from breaking and rejoining, but the segment is “flipped.” Translocations—segment of DNA breaks off and is inserted into another chromosome; can lead to duplications and deletions Figure 9.17 Chromosomal Mutations Concept 9.3 Mutations Are Heritable Changes in DNA Spontaneous mutations occur with no outside influence. • Replication errors by DNA polymerase— most are repaired but some become permanent. • Nucleotide bases can exist in 2 forms (tautomers), one common and one rare. A rare tautomer can pair with the wrong base. Figure 9.18 Spontaneous and Induced Mutations (Part 1) Concept 9.3 Mutations Are Heritable Changes in DNA • Spontaneous chemical reactions may change bases (e.g., deamination) • Errors in meiosis such as nondisjunction and aneuploidy or chromosomal breakage and rejoining. • Gene sequences can be disrupted— random chromosome breakage and rejoining can produce deletions, duplications, inversions, or translocations. Concept 9.3 Mutations Are Heritable Changes in DNA Induced mutations are caused by mutagens: • Chemicals can alter nucleotide bases (e.g., nitrous acid can cause deamination) • Some chemicals add other groups to bases (e.g., benzopyrene adds a group to guanine and prevents base pairing). Figure 9.18 Spontaneous and Induced Mutations (Part 2) Concept 9.3 Mutations Are Heritable Changes in DNA • Ionizing radiation, such as X rays, can detach electrons from atoms and form highly reactive free radicals that can change bases and break sugar phosphate bonds. • UV radiation (from sun or tanning lamps) is absorbed by thymine, causing it to form covalent bonds with adjacent nucleotides; disrupts DNA replication. Figure 9.18 Spontaneous and Induced Mutations (Part 3) Concept 9.3 Mutations Are Heritable Changes in DNA Many mutagens are naturally occurring. Plants and fungi make many chemicals for defense; some can be mutagenic, such as aflatoxin made by the mold Aspergillus. Radiation can be natural, such as UV from the sun, or human-made, such as radiation from nuclear bombs. There are about 16,000 DNA-damaging events per cell per day, of which 80% are repaired. Concept 9.3 Mutations Are Heritable Changes in DNA Mutations can have benefits: • Provides the raw material for evolution in the form of genetic diversity • Diversity may benefit the organism immediately—if mutation is in somatic cells • Mutations in germ line cells may cause an advantageous change in offspring Concept 9.3 Mutations Are Heritable Changes in DNA Mutations can be harmful if they result in loss of function of genes or other DNA sequences needed for survival. Harmful mutations in germ line cells can be passed to offspring. • If heterozygotes for the mutation mate and produce a homozygote, the mutation can be lethal. Harmful mutations in somatic cells can lead to cancer. Concept 9.3 Mutations Are Heritable Changes in DNA We try to minimize exposure to mutagens. Many things that cause cancer are mutagens. Benzopyrene is found in coal tar, car exhaust, charbroiled foods, and cigarette smoke. Public policies help reduce exposure: • Bans on cigarette smoking • International treaties banning ozonedepleting chemicals Answer to Opening Question Ancient DNA is usually destroyed in the fossilization process. But intact DNA can be found in frozen specimens and the interior of bones. PCR can amplify tiny amounts of DNA for sequencing, but samples are easily contaminated.