14 DNA STRUCTURE, REPLICATION, AND ORGANIZATION

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1 14 DNA STRUCTURE, REPLICATION, AND ORGANIZATION Chapter Outline 14.1 ESTABLISHING DNA AS THE HEREDITARY MOLECULE Experiments began when Griffith found a substance that could genetically transform pneumonia bacteria Avery and his coworkers identified DNA as the molecule that transforms rough Streptococcus to the infective form Hershey and Chase found the final evidence establishing DNA as the hereditary molecule 14.2 DNA STRUCTURE Watson and Crick brought together information from several sources to work out DNA structure The new model proposed that two polynucleotide chains wind into a DNA double helix 14.3 DNA REPLICATION Meselson and Stahl showed that DNA replication is semiconservative DNA polymerases are the primary enzymes of DNA replication Helicases unwind DNA to expose template strands for new DNA synthesis RNA primers provide the starting point for DNA polymerase to begin synthesizing a new DNA chain One new DNA strand is synthesized continuously; the other, discontinuously Multiple enzymes coordinate their activities in DNA replication Telomerases solve a specialized replication problem at the ends of linear DNA molecules DNA replication begins at replication origins 14.4 MECHANISMS THAT CORRECT REPLICATION ERRORS Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases DNA repair corrects errors that escape proofreading 14.5 DNA ORGANIZATION IN EUKARYOTES AND PROKARYOTES Histones pack eukaryotic DNA at successive levels of organization Many nonhistone proteins have key roles in the regulation of gene expression DNA is organized more simply in prokaryotes than in eukaryotes Learning Objectives After reading the chapter, you should be able to: 1. Explain the importance of the experiments of Griffith, Avery, and Hershey and Chase and how each of these experiments was integral in concluding that DNA is the genetic material. 2. Discuss the importance of Franklin s work in X-ray diffraction and Chargaff s chemical studies to understanding the 3-D structure of DNA. 3. Describe the relationship between DNA and its primary structure and how this affects secondary structure and contributes to the mechanism of DNA replication. 4. Know the key molecular events of DNA replication and how the anitparallel nature of DNA contributes to each of these events. 5. Compare and contrast DNA replication in prokaryotes and eukaryotes on each of the many levels, including enzymes related to replication, duplication of the two strands of DNA, the role of RNA in DNA replication, and the number of replication origins. Woelker 2009 DNA Structure, Replication, and Organization 118

2 6. Understand the mechanisms of DNA repair and the consequences of DNA damage that escapes repair. 7. Explain the importance of histones for DNA packaging and why these differences exist in prokaryotes and eukaryotes. 8. Understand the role of heterochromatin and nonhistone proteins in helping to regulate gene expression and apply this principle in the subsequent chapters. Key Terms deoxyribonucleic acid (DNA) bacteriophage (phage) virus transformation nucleotide nucleoside purine pyrimidine Chargaff s rules polynucleotide chain phosphodiester bond X-ray diffraction double-helix model complementary base pairing antiparallel semiconservative replication density gradient centrifugation DNA polymerase nucleoside triphosphate replication fork DNA helicase single-stranded binding proteins topoisomerase RNA primer primase discontinuous replication leading strand lagging strand DNA ligase telomeres telomerase replication origins base-pair mismatch proofreading mechanism DNA repair mechanism mismatch repair mutations chromosomal proteins chromatin histone proteins nucleosome nucleosome core particle linker 10-nm chromatin fiber 30-nm chromatin fiber solenoid chromatin euchromatin heterochromatin nonhistone proteins bacterial chromosome nucleoid plasmids Lecture Outline A. Swiss physician and chemist Friedrich Johann Miescher was the first to study the chemical composition of the cell nucleus. 1. In the spring of 1868, Miescher used pus from discarded bandages to collect white blood cells. 2. He discovered an acidic substance high in phosphorus and called it nuclein. B. DNA is the genetic material of all living organisms. 1. DNA was recognized as the hereditary molecule in DNA structure was discovered by Watson and Crick (Figure 14.1) and was key to understanding how DNA is used in a cell Establishing DNA as the Hereditary Molecule A. In the first half of the twentieth century, many scientists believed proteins were the most likely candidates for hereditary molecules; some believed nucleic acids were the hereditary molecules. B. Experiments began when Griffith found a substance that could genetically transform pneumonia bacteria. 1. Griffith could use components from dead bacteria to transform live bacteria. a. S strains of virulent bacteria were heat killed by fire. b. R strains of nonvirulent bacteria were converted to virulent bacteria when exposed to leftover components of dead S strains. 2. Transformed bacteria retained characteristics and passed them on to their offspring. a. Griffith concluded that some molecule was responsible for the transformation. b. Griffith concluded that the molecule could be passed to future generations. C. Avery and his coworkers identified DNA as the molecule that transforms rough Streptococcus to the infective form. Woelker 2009 DNA Structure, Replication, and Organization 119

3 1. Avery and his coworkers, MacLeod and McCarty, in 1944 repeated Griffith s experiments, using isolated components from the dead bacteria and enzymes to degrade those components (i.e., protein and DNA degrading enzymes). 2. Avery could inhibit transformation when DNA degrading enzymes were used and concluded that DNA was the transforming material. D. Hershey and Chase found the final evidence establishing DNA as the hereditary molecule (Figure 14.3). 1. They used bacteriophages or viruses that infect bacteria to demonstrated this. 2. Hershey and Chase radiolabeled DNA and protein components of bacteriophages and examined infected cells. 3. Only radiolabeled DNA is observed in infected cells, thus proving that DNA is the hereditary molecule DNA Structure A. Following the above experiments, a highly competitive scientific race to discover the structure of DNA began. B. Watson and Crick brought together information from several sources to work out DNA structure. 1. Previous research deciphered the building blocks of DNA. a. DNA is made of four possible types of nucleotides. b. Each nucleotide is made of a five-carbon sugar, a phosphate group, and one of four nitrogenous bases (adenine, thymine, guanine, or cytosine). (1) Adenine and guanine are purine nucleotides. (2) Thymine and cytosine are pyrimidine nucleotides. 2. Chargaff discovered that purine and pyrimidine bases occur in equal ratios. a. Adenine equals thymine. b. Guanine equals cytosine. 3. Other research determined DNA contains nucleotides joined to form polynucleotide chains (Figure 14.4). a. Nucleotides are oriented such that the 5' OH group on one is attached to the 3' OH group on another with the phosphate group in between (i.e., sugar phosphate sugar). b. Nucleotides are held together by strong phosphodiester bonds. 4. Wilkins and Franklin used X-ray diffraction techniques to examine the arrangement of molecules in DNA (Figure 14.5). a. Patterns indicated that DNA molecules were about 2 nm in diameter. b. Patterns also indicated that DNA is helical in shape. C. The new model proposed that two polynucleotide chains wind into a DNA double helix (Figure 14.6). 1. Their model proposed DNA was actually two strands connected by the nitrogen base component of opposing nucleotides (hydrogen bonds). a. Based on size constraints, A T and G C pairs on opposing strands fit the model of likely complementary pairing (as suggested by Chargaff s data). b. Similarly, the two strands of nucleotides fit properly when oriented antiparallel to one another (i.e., 3' end of one strand opposite the 5' end of the other strand). 2. The two strands twist around one another to form a right-handed double helix. a. The strands are held by hydrogen bonds. b. Each base pair occupies 0.34 nm of space in the length of the strands, and ten base pairs occupy the length before a turn of the helix (every 3.4 nm). 3. Watson and Crick realized the blueprint held within DNA. a. The only difference in DNA strands are the nitrogenous bases (A, T, G, C) of the nucleotides. b. Watson and Crick proposed that the sequence of nucleotides in a DNA strand might be the code for our traits DNA Replication A. Data on DNA structure was used to suggest how DNA replicates. 1. Base pair differences (sequences) appeared key to the code in DNA. 2. Watson and Crick suggested that the key to replication had to do with repeating the nucleotides while somehow retaining the original sequence. a. Suggested strands must somehow unwind to reveal the sequence of nucleotides. b. Each strand serves as template for replication. Woelker 2009 DNA Structure, Replication, and Organization 120

4 B. Semiconservative replication is suggested as a method of DNA replication (Figure 14.7). 1. Watson and Crick suggested that each DNA strand is maintained and used as a template to make two new strands of DNA. a. In this method, an old DNA strand then becomes paired with a newly made DNA strand. b. In other suggested methods, DNA strands may have all old or new DNA strands combining (conservative replication) or a mosaic of old and new strands (dispersive replication) (Figure 14.8). C. Meselson and Stahl showed that DNA replication is semiconservative (Figure 14.9). 1. A heavy DNA double helix was made using nucleotides containing all N 15 molecules. a. The heavy DNA was then allowed to replicate in medium containing only nucleotides containing light (i.e., N 14 ) nucleotides. b. Centrifugation of the resulting DNA strands demonstrated that an original strand ends up attached to a new strand following replication. D. DNA polymerases are the primary enzymes of DNA replication. 1. DNA polymerases use nucleoside triphosphates (datp, dgtp, dctp, or dttp) as substrates for making new DNA strands. 2. The polymerase recognizes the nucleotide on the template strand and positions the proper complementary nucleoside triphosphate in the growing new strand. 3. The polymerase can only add a nucleotide triphosphate to the 3' OH end of an existing nucleotide strand (Figure 14.10). 4. The key molecular events of DNA replication are: a. Two strand of DNA molecule unwind for replication to occur. b. Nucleotides are added only to an existing chain. c. The overall direction is from the 5' to the 3' direction d. Nucleotides add according to the A T and G C base pairing rules. E. Helicases unwind DNA to expose template strands for new DNA synthesis (see Table 14.1 for a list of major enzymes of replication). 1. DNA helicase helps unwind the DNA strands exposing the nucleotide bases (Figure 14.12). 2. Single-stranded binding proteins stabilize the open DNA strands for the replication process. 3. Topoisomerases help undo any twisting that occurs in the DNA strands as they are unwound. F. RNA primers provide the starting point for DNA polymerase to begin synthesizing a new DNA chain 1. A primase enzyme adds the first nucleotide (primer) to the new strand. a. The primer is a short strand of RNA. b. The primer is later removed and replaced by DNA before replication is complete. G. One new DNA strand is synthesized continuously; the other discontinuously. 1. DNA polymerases can catalyze the synthesis of one strand continuously (i.e., in the 5' to 3' direction, which is considered the leading strand). 2. Because of its orientation, the other strand is replicated in pieces or discontinuously (Figure and Figure 14.12). a. The polymerase must unattach and reattach as it replicates this strand (lagging strand). b. The short DNA fragments added are referred to as Okazaki fragments. H. The multiple enzymes coordinate their activities in DNA replication (Figure 14.12). 1. The helicase unwinds the DNA strands first. 2. The primases then lay down the first primers at a site called the replication fork. 3. DNA polymerase then starts laying down other nucleotides. 4. If the polymerase reaches a site where a primer is, the polymerase leaves, and another special polymerase binds the growing strand to remove the RNA primer. 5. DNA ligase helps close any nicks in the DNA strands once the primers are removed. I. Telomerases solve a specialized replication problem at the ends of linear DNA molecules. 1. When starting replication at the end of a linear piece of DNA, removal of the primer results in a gap, which normal DNA polymerases are unable to fix (Figure 14.13). a. Over time, linear pieces of DNA will get shorter following each replication. b. Eukaryotes have a special polymerase (telomerase) that adds nucleotides to the end of DNA before replication (Figure 14.14). 2. Then ends of eukaryotic DNA are referred to as telomeres since they usually have repeated DNA that does not typically code for proteins. Woelker 2009 DNA Structure, Replication, and Organization 121

5 a. Eukaryotic cells typically have a finite number of mitotic divisions they can undergo before they stop dividing and die. b. Telomerase activity may be linked to aging (underactive enzymes) and cancer (overactive enzymes). J. DNA replication begins at replication origins. 1. Replication can occur at multiple sites (or forks) on a chromosome (Figure 14.15). 2. The replicating forks meet as the replication is complete Mechanisms That Correct Replication Errors A. Most of the mistakes that occur, called base-pair mismatches, are corrected. B. Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases. (Figure 14.17). 1. Kornberg and Brutlag described the ability of polymerases to work only if bases are paired correctly. 2. Error rates due to DNA polymerase mismatching is low (1 in 10 6 ). C. DNA repair corrects errors that escape proofreading (Figure 14.17). 1. Mismatch repair systems search DNA for distortions that may be associated with incorrect base pairings. 2. The mismatch is cut out and repaired with a DNA polymerase and a DNA ligase. a. Faulty mismatch systems are associated with human disorders (xeroderma pigmentosum). b. Differences that do not get fixed and persist in DNA are referred to as mutations. Insights from the Molecular Revolution: A Fragile Connection between DNA Replication and Mental Retardation A. Fragile X syndrome results from breaks that occur on the X chromosome. 1. Common effects of the syndrome are mental retardation, facial abnormalities, and enlarged testes in males. 2. The condition is inherited and appears in males more than females. B. Investigators have examined the occurrence of the condition from generation to generation. 1. Molecular probes examining the region usually damaged on the X chromosome have revealed that the DNA contains several triplet repeats of nucleotides (CCG) in affected individuals. 2. The triplet repeat of nucleotides varies with more repeats associated with more effects of the disease. a. Normal people have 6 50 copies of the repeat sequence. b. Mildly affected people have copies of the repeat sequence, and seriously affected individuals have copies. C. Fragile X syndrome is likely associated with overreplication problems. 1. Increase in CCG triplet may inhibit the expression of other important genes (e.g., FMR-1 gene). 2. Methylation of CCG and nearby sites on DNA may be involved in inhibiting expression of other genes DNA Organization in Eukaryotes and Prokaryotes A. Enzymatic proteins are the essential catalyst for every step in DNA replication. B. These proteins are known collectively as chromosomal proteins. C. The single DNA molecule of a prokaryotic cell is more simply organized and has fewer associated proteins. D. Histones pack eukaryotic DNA at successive levels of organization. 1. In eukaryotes, DNA is associated with nonhistone and histone proteins. a. DNA, histones, and nonhistones are referred to as chromatin. b. A single DNA strand with all its associated histones and nonhistones is a chromosome. 2. Prokaryotes have fewer molecules associated with their DNA. E. Histones and DNA Packing. 1. Histones use their positive charge to interact with negatively charged DNA. a. There are five different types of histones (H1, H2A, H2B, H3, H4). b. Histones affect DNA packaging and expression. 2. At its simplest level, histones 2A, 2B, 3, and 4 form a core for DNA to wrap around, forming a structure called a nucleosome (Figure 14.18). a. The diameter of the nucleosome gives the structure its name (10-nm chromatin fiber). F. Histones and Chromatin Fibers. 1. Histone H1 helps link multiple nuclesomes in a solenoid-like structure called a 30-nm chromatin fiber. a. About six nucleosomes per turn make up a solenoid. Woelker 2009 DNA Structure, Replication, and Organization 122

6 b. DNA is harder to degrade when wrapped by histones. G. Packing at Still Higher Levels: Euchromatin and Heterochromatin. 1. Loosely packaged regions of a chromosome are called euchromatin. 2. Densely packaged regions of a chromosome are called heterochromatin. a. Histone H1 may be involved in packaging of heterochromatin. b. Inactive X chromosomes in females (Barr body) are a form of complete chromosome packaging. H. Many nonhistone proteins have key roles in the regulation of gene expression. 1. DNA must be accessible for gene expression. 2. Many nonhistone proteins affect DNA packaging by affecting the ability of histones to package DNA. 3. Other nonhistone proteins directly affect gene expression or are themselves components required to control gene expression. I. DNA in prokaryotes is organized differently compared to eukaryotes. 1. Most bacteria have a single circular piece of DNA (chromosome). a. Replication begins in a single region (origin) moving in two forks around the circular DNA (Figure 14.19). 2. The chromosome is packed into an irregular mass (nucleoid) in the cytoplasm. a. Small extrachromosomal DNA molecules called plasmids can be found in the cytoplasm. Woelker 2009 DNA Structure, Replication, and Organization 123