Presentation on theme: "11 Announcements Chapter 10 Online Quiz Deadline: Midnight If you have not yet picked up your graded Exam #1, it is on the front desk. BIO 122 is a prerequisite."— Presentation transcript:
11 Announcements Chapter 10 Online Quiz Deadline: Midnight If you have not yet picked up your graded Exam #1, it is on the front desk. BIO 122 is a prerequisite for BIO/HON 214: Bioterrorism & Emerging Infectious Diseases (Spring Semester).
11 DNA: The Genetic Material The Structure of DNA Determining the DNA Replication Mechanism The Molecular Mechanisms of DNA Replication DNA Proofreading and Repair Practical Applications of DNA Replication
11 DNA: The Genetic Material? The Question: WHAT MOLECULE CARRIES GENETIC INFORMATION? By the early 1900’s, geneticists had associated the presence of genes with chromosomes. Circumstantial evidence pointed to DNA as the genetic material, though protein was a strong contender. DNA, as was protein, was found in the nucleus and chromosomes, which were already known to carry genes. A dye that binds to DNA showed that the amount of DNA in somatic cells (diploid) was twice that in eggs or sperm (haploid), as would be expected from Mendel’s discoveries.
11 DNA: The Genetic Material? In the 1920s, the English physician Frederick Griffith did experiments with two strains of Streptococcus pneumoniae. He discovered that a chemical transforming principle from one strain could could cause a heritable change in the other strain. http://genome.microbio.uab.edu/strep/info/strep5.gif http://web.indstate.edu/ thcme/micro/quell.gif Polysaccharide Capsule
11 DNA: The Genetic Material? Two strains of Streptococcus pneumoniae: S strain (smooth colonies): with polysaccharide capsule protected from host’s immune system causes pneumonia in host (e.g., mouse) virulent (causes disease) R strain (rough colonies): without polysaccharide capsule not protected from host’s immune system host does not become ill non-virulent strain http://web.indstate.edu/ thcme/micro/quell.gif
Figure 11.1 Genetic Transformation of Nonvirulent Pneumococci
11 DNA: The Genetic Material? Oswald T. Avery and colleagues spent several years identifying the transforming principle. They finally published the work in 1944. They treated the extract from the S- strain bacteria in various ways to destroy different types of substances (e.g., proteins, nucleic acids, carbohydrates, or lipids) but retain others. When DNA was destroyed, the transforming activity was lost, but when DNA was left intact, the transforming activity survived.
11 DNA: The Genetic Material In 1952, Alfred D. Hershey and Martha Chase performed experiments confirming that DNA is the genetic material. The T2 bacteriophage, a virus that attacks E. coli, consists almost entirely of a DNA core packed in a protein coat. When a T2 bacteriophage attacks a bacterium, part but not all of the virus enters the bacterial cell. The Hershey-Chase experiment determined which part of the virus (protein or DNA) entered the bacterium.
Figure 11.2 T2 and the Bacteriophage Reproduction Cycle (Part 1)
Figure 11.2 T2 and the Bacteriophage Reproduction Cycle (Part 2)
11 DNA: The Genetic Material Some viruses were labeled with radioactive sulfur, which is present in proteins but not in DNA. Other viruses were labeled with radioactive phosphorus, which is present in DNA but absent from most proteins. The labeled sulfur (and thus the viral protein) separated from the bacteria, but the labeled phosphorus (and thus the viral DNA) remained with the bacteria.
11 The Structure of DNA THE NEXT SET OF QUESTIONS… Scientists set out to determine the structure of DNA hoping to find the answers to two questions: How is DNA replicated between nuclear divisions (remember the S Phase)? DNA REPLICATION How does DNA cause the synthesis of specific proteins? PROTEIN SYNTHESIS The structure of DNA was determined after many types of evidence were combined.
Figure 11.4 X-Ray Crystallography Revealed the Basic Helical Structure of the DNA Molecule Rosalind Franklin and Maurice Wilkins were able to provide key information about the structure of DNA based on X-ray crystallography. Pattern suggests the placement of atoms in a crystal. http://www.newhumanist.org. uk/images/0209/franklin.jpg Rosalind Franklin
11 The Structure of DNA OTHER INFORMATION: By the 1950s it was known that DNA was a polymer of nucleotides. The four nucleotides that make up DNA differ only in their nitrogenous bases. There are two purines (adenine and guanine) and two pyrimidines (cytosine and thymine) present in DNA. In 1950, Erwin Chargaff noted that in DNA from all species tested, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine. A = T G = C suggesting pairing of these bases.
11 The Structure of DNA American geneticist James D. Watson (left) and English physicist Francis Crick (right) established the general structure of DNA in 1953. http://salmon.psy.plym.ac.uk/year3/PSY339 EvolutionaryPsychologyroots/watson-crick-dna.jpg
Figure 11.6 DNA Is a Double Helix The DNA molecule is a double- stranded helix. The diameter of the DNA molecule is uniform. The twist in DNA is right-handed. The two strands run in different directions (they are antiparallel). The sugar–phosphate backbones of each strand coil around the outside of the helix. The nitrogenous bases point toward the center of the helix. Hydrogen bonds between complementary bases hold the two strands together. A always pairs with T (two hydrogen bonds). G always pairs with C (three hydrogen bonds).
11 The Structure of DNA The phosphate groups link the 3 carbon of one deoxyribose molecule to the 5 carbon of the next. A single strand of DNA has a 5 phosphate group at one end (the 5 end) and a free 3 hydroxyl group at the other end (the 3 end). Recall the structure of a nucleotide and polynucleotide: http://www.bio.miami.edu/dana/250/nucleotides.jpg
Figure 11.7 Base Pairing in DNA Is Complementary
11 The Structure of DNA NEXT QUESTION: How does DNA replicate? In Watson & Crick’s 1953 publication in Nature, they stated: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” T ---- A C ---- G
11 Determining the DNA Replication Mechanism American biochemist Arthur Kornberg demonstrated that the DNA molecule contains the information needed for its own replication. Kornberg showed that DNA can replicate in a test tube with only a specific enzyme (DNA polymerase) and a mixture of four precursors (deoxyribonucleoside triphosphates): dATP, dCTP, dGTP, and dTTP. http://www.bio.miami.edu/dana/250/nucleotides.jpg Arthur Kornberg & Francis Crick (1980) http://profiles.nlm.nih.gov/SC/B/B/F/K/_/scbbfk.jpg
Figure 11.8 Three Models for DNA Replication Semiconservative Conservative Dispersive Model
11 Determining the DNA Replication Mechanism Theoretically, DNA could serve as its own template in one of three different ways: Semiconservative replication would use each parent strand as a template for a new strand. Conservative replication would build an entirely new double helix based on the template of the old double helix. Dispersive replication would use fragments of the original DNA molecule as templates for assembling two molecules.
11 Determining the DNA Replication Mechanism Matthew Meselson and Franklin Stahl demonstrated in 1957 that DNA replication is semiconservative by using a technique called density labeling. They used DNA labeled with “heavy” nitrogen ( 15 N). http://education.jlab.org/qa/atom_model_03.gif
11 The Molecular Mechanisms of DNA Replication How does DNA replication take place? The hydrogen bonds between the two strands are broken, making each strand available for base pairing. The new nucleotides are covalently bonded to each growing strand.
11 Announcements Be sure to sign (signature) attendance sheet. Dr. Faivre (BIO 122 professor): Announcement: School for Field Studies
11 Semiconservative Model Recall the Semiconservative model of DNA replication: original strands separate and complimentary strands are produced for each of the original strands to form two new DNA molecules. [Meselson-Stahl Experiment]
11 The Molecular Mechanisms of DNA Replication SO, HOW ARE THE NEW STRANDS PRODUCED? 1) The hydrogen bonds between the two original strands are broken, making each strand available for base pairing. 2) The new nucleotides are covalently bonded to each growing strand.
11 Where DNA Replication Begins Modified: http://web.wtez.net/n/s/ns54007/gene/gene-jpeg/origin.jpg Large linear chromosome: Multiple (100’s) origins of replication Small circular chromosome: Single origin of replication Modified: http://www.langara.bc.ca/ biology/mario/Assets/OriginRep.jpg
Figure 11.15 Many Proteins Collaborate at the Replication Fork
11 Key Player in DNA Replication – DNA Polymerase DNA polymerase III is shaped like a hand, and in the side-on view, its “fingers” can be seen curling around the DNA. These “fingers” can recognize the different shapes of the four bases (white; the DNA “backbone” is shown in red). -Purves et al.
Figure 11.10 Each New DNA Strand Grows from its 5 End to its 3 End
11 Applications: PCR & DNA Sequencing Applications! Polymerase Chain Reaction DNA Sequencing BOTH ARE BASED ON THE PROCESS OF DNA REPLICATION. http://media.biocompare.com/bcimages/items/256700.jpghttp://www.ctegd.uga.edu/lab_pages/images/sequencer.jpg
11 Practical Applications of DNA Replication The polymerase chain reaction (PCR) technique is a simple method for making multiple copies of a DNA sequence. Having lots of DNA helps scientists do the things they need to do in the laboratory! PCR cycles through three steps (recall DNA replication): DENATURATION: Double-stranded fragments of DNA are heated to denature them into single strands. (~ helicase unraveling the double helix) ANNEALING: A short primer is added, along with the four dNTPs. (~ primase adding a primer) EXTENSION: DNA polymerase catalyzes the production of new DNA strands.(~ DNA polymerase adding bases)
11 Practical Applications of DNA Replication PCR did not become practical until the discovery of a DNA polymerase that could survive the heat required to denature the DNA. Such a DNA polymerase was found in bacteria that live in hot springs at Yellowstone National Park. The biochemist Kerry Mullis earned a Nobel prize for this technique. http://www.travel247365.com/photos/yellowstone/yellowstone017.jpghttp://www.pcrstation.com/images/mullis.jpg
11 Practical Applications of DNA Replication The technique of DNA sequencing hinges on the use of modified nucleotides (ddNTPs). dNTPs contain the sugar 2-deoxyribose. ddNTPs contain the sugar 2,3-dideoxyribose. Like dNTPs, ddNTPs are picked up by DNA polymerase and added to a growing DNA chain. ddNTPs lack a hydroxyl group at the 3 position, however, so no new nucleotide can be added after a ddNTP, and synthesis ends.
11 Practical Applications of DNA Replication Sequencing begins by denaturing a fragment of DNA. The single-stranded DNA is mixed with DNA polymerase, short primer strands, the four normal dNTP substrates, and small amounts of the four ddNTPs, each with a fluorescent tag. In solution, DNA polymerase synthesizes strands of DNA using mostly the normal dNTP substrates. When DNA polymerase encounters a ddNTP, chain growth stops. The result is a solution with template DNA strands and shorter complementary strands, each one ending with a fluorescently tagged ddNTP.
11 Practical Applications of DNA Replication The new strands are denatured from the templates and separated by electrophoresis, a technique that separates strands by length. The shortest fragments should be one base longer than the primer strand. The color of the fluorescent tag at the end of this sequence indicates the type of ddNTP that was added. If this was ddATP, for example, then the first base on the template strand (after the primer sequence) is T. The remainder of the bases on the template strand can be determined in a similar manner.
11 Output from a DNA sequencer. What you see is an electropherogram. http://www.niams.nih.gov/rtbc/184.108.40.206/imagestore/test/images/ab/rag/rag2.jpg
11 More Information Beyond Summary slides that support the graphics discussed previously…
11 The Molecular Mechanisms of DNA Replication In DNA replication, nucleotides are added to the 3 end of the growing strand. The three phosphate groups of the deoxyribonucleoside triphosphate are attached to the 5 position of the sugar. Energy for synthesis of nucleotides to the growing chain comes from breaking the bonds between these three phosphates.
11 The Molecular Mechanisms of DNA Replication A huge protein complex catalyzes DNA replication. This replication complex recognizes an origin of replication on a chromosome. DNA replicates in both directions from the origin, forming two replication forks. In DNA replication, both strands of DNA act as templates. Recent evidence suggests that the replication complex is stationary, and DNA threads through it.
11 The Molecular Mechanisms of DNA Replication The enzyme DNA helicase uses energy from ATP to unwind the two DNA strands. Special proteins bind to the unwound strands to keep them apart. Small chromosomes, such as those found in bacteria, have a single origin of replication. Replication in bacteria produces two interlocking circular DNAs that are separated by the enzyme DNA topoisomerase. Large chromosomes can have hundreds of origins of replication. Replication occurs at many different sites simultaneously.
11 The Molecular Mechanisms of DNA Replication DNA polymerases cannot build a strand without having an existing strand, called a primer, to start from. In DNA replication, the primer strand is a short strand of RNA complementary to the DNA template strand. An enzyme called a primase makes the primer strand.
11 The Molecular Mechanisms of DNA Replication Most cells contain more than one DNA polymerase. Only one of the polymerases is responsible for chromosomal DNA replication. The others are involved in primer removal and DNA repair.
11 The Molecular Mechanisms of DNA Replication Recall that new bases are always added to the 3 end of a growing DNA strand. The strands in the template DNA are antiparallel, however. As a result, as the strands pass through the replication complex, one strand (the leading strand) will be in the correct orientation for addition of new nucleotides. The other strand (the lagging strand) will be in the reverse orientation.
Figure 11.16 The Two New Strands Form in Different Ways
11 The Molecular Mechanisms of DNA Replication Because of its backward orientation, the lagging strand must grow in relatively small, discontinuous pieces, called Okazaki fragments. Each Okazaki fragment requires an RNA primer strand, which is formed by RNA primase. DNA polymerase III synthesizes complementary DNA starting from the 3 end of the new primer and working toward the previous Okazaki fragment.
11 The Molecular Mechanisms of DNA Replication When DNA polymerase III reaches the previous Okazaki fragment, it is released. DNA polymerase I then replaces the RNA primer of the previous Okazaki fragment with DNA. Finally, DNA ligase catalyzes formation of the phosphodiester linkage that joins the two Okazaki fragments.
11 The Molecular Mechanisms of DNA Replication Recall that replication of the lagging strand occurs by the addition of Okazaki fragments to RNA primers. Beyond the very end of a linear DNA molecule, there is no place for a primer to bind. New chromosomes formed after DNA replication have single-stranded DNA at each end. This single-stranded region is cut off, slightly shortening the chromosome after each cell division.
11 The Molecular Mechanisms of DNA Replication Many eukaryotic chromosomes have repetitive sequences called telomeres at their ends that shorten after each round of cell division. After a given number of cell divisions, the telomeres have shortened to the extent that they are no longer able to stabilize the ends of the chromosomes, and no cell division can occur. This results in cell death and explains in part why cells do not last the entire lifetime of the organism.
11 The Molecular Mechanisms of DNA Replication Constantly dividing cells, such as bone marrow, germ line, and more than 90 percent of cancer cells, produce an enzyme called telomerase that catalyzes the addition of any lost telomeric sequences.
11 DNA Proofreading and Repair Although errors in DNA replication (mutations) are essential for evolution, the vast majority of DNA errors are neutral at best and fatal at worst. To minimize the number of errors, our cells have three DNA repair mechanisms: Proofreading Mismatch repair Excision repair
11 DNA Proofreading and Repair As they add new bases to a growing strand, DNA polymerases make a proofreading check. When a DNA polymerase recognizes an error, it removes the wrong nucleotide and tries again. The error rate of DNA polymerase on each attempt is only about 1 in 10,000. This proofreading function reduces the overall error rate to about one base in a billion.
11 DNA Proofreading and Repair The mismatch repair mechanism scans new DNA for mismatched base pairs. The mismatch repair mechanism operates before the new DNA strand is methylated. This mechanism can distinguish between the methylated template strand and the unmethylated new strand. Thus, this mechanism can determine which base is correct (the base on the template strand) and which base needs to be replaced.
11 DNA Proofreading and Repair Excision repair proteins operate over the life of a cell. DNA is subject to damage by chemicals, radiation, and random spontaneous chemical reactions. Excision repair enzymes “inspect” the cell’s DNA for damage, then cut the damaged strand and remove it. DNA polymerase and DNA ligase fill in and seal up the resulting gap.