Chapter 9: DNA and its role in Heredity PDF

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This note covers concepts for chapter 9: DNA and its role in heredity. ...

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Scientists used two types of evidence to show that DNA is the genetic material: circumstantial and experimental. However, scientists rely on experiments to provide evidence of a cause and effect relationship. Recall that chromosomes in eukaryotic cells contain DNA, but they also contain proteins that are bound to DNA. Therefore, it was difficult to rule out the possibility that genetic information might be carried in proteins. Biologists used model organisms such as bacteria in transformation experiments, and they found that the addition of DNA from one strain of bacterium could genetically transform another strain. # # Researches to support that the genetic material of a cell is DNA: 1. Miescher (1868): isolated "nuclein" from cells (which didn't make connection to genetics)! 2. Feulgen (1924): DNA staining- he used stain to pinpoint where DNA is in a cell and concentration $ $ $ $ $ of DNA in a cell. The stain is a dye that turns bright purple when it reacts with $$$$$$$ $ $ $ $ DNA. # 3. Mirsky (late 1920s): looked at DNA content of cells from various organisms (chicken, cow, frog,# $ $ $ $ and human). He found that regardless of the type of organisms, gametes such as # $ $ $ $ eggs and sperms each have half the DNA content of tissue cells. # 4. Avery Macleod McCarty (1944): demonstrates that DNA is where genetic information is stored. # 5. Chase & Hershey (1952): $ DNA is the material of inheritance. # 6. Watson, Crick, Wilkins, Franklin (1953): Franklin's work in X-ray diffraction was important in# $ $ $ $ revealing the structure of the DNA molecule. Watson and Crick reveals the double# $ $ $ $ helix structure of DNA with the help of Franklin's work. # # Transforming Principle (1928): One of the most famous biology experiments of the 20th century, which eventually lead to our understanding that DNA was the molecule of inheritance genetics. # # # # # # # # # # # # # # # # Rough (R) Strain: does not have # capsule. Therefore, susceptible to attack by host immune system. # Avirulent. ! # ! # Smooth (S) Strain: has capsule made # of polysaccharide. Capsule protects # bacteria from host immune system. Virulent. # # # # # # #

Components of DNA and their arrangements: 1. Component molecules: the DNA molecule is composed of three types of component molecules such as: phosphate groups, the sugar deoxyribose, and the bases adenine, thymine, guanine, and cytosine (A, T, G, C) ! 2. Nucleotides: These three molecules (phosphate, sugar, and bases) link to form the basic building block of DNA, nucleotides. Each nucleotide is composed of one sugar, one phosphate group, and one of the four bases. ! 3. The double helix: The sugar from one nucleotide links with the phosphate from the next to form the handrails of the double helix. Meanwhile, the bases form the stair-steps. Each base extending across the helix to link with a complementary base extending from the other side. ! # # # # # # # # # Base pairs in DNA can interact with other molecules:

Base pairs of anti-parallel DNA strands are held together via HYDROGEN BONDS.!

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Formation of a nucleic acid - " Phosophodiester bond" ! 1. New nucleotides are added to an existing chain one at a time.! 2. Pentose sugar in the LAST. Existing chain and the phosphate on the NEW nucleotide undergo condensation reaction. ! 3. Bond is formed by adding the 5 prime phosphate end of the new nucleotide to the 3 prime end of the nucleic acid. ! 4. Nucleic acids are grown in the 5' to 3' direction. ! Structure of DNA: ◦ Sugar and phosphate groups form the backbones in the double helix.! ◦ The presence of a major and a minor groove:! ‣ Major/minor groove is produced by twisting ! ‣ Backbones not evenly spaced relative to one another! ‣ Allows proteins to contact bases! • This is necessary for replication and transcription ! ◦ The two stands of DNA in the helix are "anti-parallel"! ‣ 5' to 3'! ‣ 3' to 5'

DNA synthesis requires energy: ◦ The raw materials for DNA synthesis are nucleotides (dNTPs):! ‣ Deoxyadenosine triphosphate (dATP)! ‣ Deoxythymidine triphosphate (dTTP)! ‣ Deoxycytidine triphosphate (dCTP) ! ‣ Deoxyguanosine triphosphate (dGTP)! ◦ DNTPs each carry three phosphate groups, during DNA synthesis, the two outer phosphate groups are released in an exergonic reaction, so that the final nucleotide is a monophosphate (adenine, thymine, cytosine, or guanine). The release of the two outer phosphate groups provides energy for the formation of a phosphodiester bond between the single remaining phosphate group of the incoming nucleotide and the 3' carbon on the sugar at the end of the DNA chain.

Chapter 9.2: DNA replicates semiconservatively Genetic material is able to replicated both completely and accurately during the cell cycle.# • Mechanism of DNA replication is known as "semi-conservative". ! • Brief Steps of replication: ! ◦ The 2 strands of the DNA double helix are separated at an "ori". ‣ A.K.A: The hydrogen bonds holding the bases together are temporarily broken. ! ◦ Each strand is a template by which a daughter strand is synthesized. ! ◦ The daughter strands remain attached to their template strand.! DNA replication: # 1. DNA replication begins with the binding of a large protein complex (the pre-replication complex) ! 2. Chromosomes have one region "ori" (origin of replication) to which the pre- replication complex binds.! 3. Once the pre-replication complex binds to it, the DNA double helix unwinds and replication proceeds in both directions around the circle, forming the replication forks. ! 4. As the DNA opens, two Y-shaped structures called replication forks are formed, together making up what's called a replication bubble. The replication forks will move in opposite directions as replication proceeds. ! 5. The enzyme helicase is present to separate the two strands in a DNA double helix (unwinds the helix). When the double helix structure is separated, it makes them available for new base pairing. This enzyme helicase breaks hydrogen bonds between bases on the two strands and separating them (move the replication forks forward)! 6. The enzyme DNA Polymerases add nucleotides to the 3' end of the NEW DNA strand with the help of a primer. Primer is a short single strand of RNA. The primer is complementary to the DNA template and is synthesized ONE nucleotide at a time by an enzyme known as primase. ! 7. Proteins called single-strand binding proteins coat the separated strands of DNA near the replication fork, keeping them from coming back together into a double helix (keep the unpaired template strands apart during replication). ! 8. The enzyme Gyrase/ Topoisomerase remove or create supercoiling in duplex DNA! 9. As newly added nucleotides form complementary (A with T, and G with C) base pairs with template DNA, they are covalently linked together by phosphodiester bonds whose base sequence is complementary to the bases in the template strand. Replication enzymes read the template DNA in the 3' to the 5' direction. ! 10. DNA polymerase add nucleotides to the 3' end of growing strand; complementary to the old template.! 11. Because DNA replication happens really fast, DNA polymerase needs help to hold to the DNA as it zooms along sliding clamp binds to DNA/ DNA polymerase complex. ! Primer is needed to start DNA replication: ! " ◦ DNA polymerase cannot lay down the first nucleotide, it needs the new strand to be primed first. ! " ◦ The primer is made up of RNA and is complementary to the DNA template (3-10 nucleotides).

Sliding clamp plays a key role in DNA replication: ◦ Increases efficiency of DNA polymerase processes ! ◦ Ensures polymerase is in the correct orientation ! ◦ Sliding clamp is the binding bite for:! ‣ DNA ligase ! ‣ Methylation enzymes ! ‣ DNA repair enzymes !

# # # # # Two bonds are made when a nucleotide is added to the new strand: 1. Covalent phosphodiester bond with the new strand ! 2. Hydrogen bonds between the template G and the new C ! ! The two new strands form in different ways: as the parent DNA unwinds, both new strands are synthesized in the 5'- to 3' direction, although their template strands are antiparallel. The leading strand grows continuously forward, but the lagging strand grows in short, discontinuous stretches called Okazaki fragments. Okazaki fragments in eukaryotes are 100 to 200 nucleotides long. ! 1. Leading strand: oriented so that it can grow continuously at its 3' end as the fork opens up. "Grows FORWARD"! 2. Lagging strand: must be synthesized differently because it grows in the direction away from the replication fork. "BACKWARD"! ◦ Requires the synthesis of relatively short, discontinuous stretches of sequence of nucleotides. ! ◦ These discontinuous stretches are synthesized just as the leading strand is, by the addition of new nucleotides one at a time to the 3' end, but the new strand grows away from the replication fork --> Okazaki fragments. !

# The lagging strand story: ! 1. A primer is needed to start the synthesis of the leading strand, but each Okazaki fragment requires its own primer to be synthesized by the primase. ! 2. DNA polymerase then synthesizes an Okazaki fragment by adding nucleotides to one primer until it reaches the primer of the previous fragment. ! 3. At this point, a different DNA polymerase removes the old primer and replaces it with DNA.! 4. Currently, the final phosphodiester linkage between the adjacent Okazaki fragments is MISSING.! 5. The enzyme DNA ligase catalyzes the formation of that bond, linking the fragments and making the lagging strand WHOLE / complete. ! ! ! ! ! ! !

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The 3' to 5' strand (OLD) determines what base will be added to the newly synthesized 5' to 3' strand. In this case, the template has a G, which means DNA polymerase will add a C to the new strand.

Telomeres: are strings of repetitive sequences at the ends of chromosomes to prevent chromosomes from joining and to maintain integrity of chromosome. Telomeres act as caps that protect the internal regions of the chromosomes. # " Characteristics of telomeres: ‣ Progressively shorten as we age.! • Length is associated with aging, cancer, and cell differentiation! ‣ Facilitate pairing of homologous chromosomes ! ‣ Chromosomes without telomeres resemble double stranded DNA breaks ! • Repair proteins attempt to repair "break" by ligating 2 chromosomes together ! # Telomerase is an enzyme that prevents telomere from shortening. # ◦ This enzyme is an RNA dependent DNA polymerase, meaning that this enzyme can make DNA using RNA as a template. ! ◦ This enzyme binds to a special RNA molecule that contains a sequence complementary to the telomeric repeat. ! ◦ MOREOVER, this enzyme extends by adding nucleotides to the overhanging strand of the telomere DNA using this complementary RNA as a template. ! ◦ When the overhanging strand is long enough, a matching strand can be made by the normal DNA replication machinery to produce double stranded DNA (with the help of RNA primer and DNA polymerase). ! # # • This enzyme is usually not active in most somatic cells, instead they are active in germ cells (cells that make sperm and eggs), and stem cells. Those are the cells that need to undergo many cell divisions. ! • Telomerase is also active in cancer cells. If telomerase could be inhibited by drugs as part of cancer therapy, the growth of cancerous tumor could be stopped because without telomerase, these cells have relatively short lifespan. ! - Allows primase and DNA polymerase to extend 5' # ends.! # - Does NOT repair the new 3' end "overhangs". # # # # # # # # # # # # #

End- replication problem: DNA at the ends of chromosomes cannot be fully copied in each round of replication, which results in slow/gradual shortening of the chromosome. # ◦ When the replication fork reaches the end of the chromosome, however there is a short stretch of DNA that does not get covered by an Okazaki fragment. Importantly, there's no way to get the fragment started because the RNA primer would fall beyond the chromosome end. ! ◦ The primer of the last Okazaki fragment that does get made, CANNOT be replaced with DNA like other primers. ! ◦ LAST RNA primer removed --> no 3' end OH to extend! # # # # # # # # # # # # # # # # # # Errors in DNA replication can be repaired: most errors can be repaired in DNA replication through two major repair mechanisms: # 1. Proofreading: occurs right after DNA polymerase inserts a nucleotide. When a DNA polymerase recognizes a mispairing of bases, it removes the improperly introduced nucleotide and tries again. ! A. DNA polymerase proofreads each NEW nucleotide with the template. ! B. Removal of improperly introduced nucleotide is known as: Exonuclease activity

2. Mismatch repair: occurs after DNA has been replicated. A second set of proteins surveys the newly replicated molecule and looks for mismatched base pairs that were missed in proofreading. # ◦ A portion of the DNA including the incorrect nucleotide is removed, and then a DNA polymerase inserts the correct sequence. ! # • Also, proteins that are made during S-phase: can scan newly replicated DNA molecules for mismatch errors and repair these errors.! # • DNA polymerase adds correct # bases. ! # • DNA ligase repairs nick. # # # #

Summary of DNA replication: • Replication begins at special sites known as origins of replication (Ori) ◦ Prokaryotes: 1 ori per chromosome ! ◦ Eukaryotes: 100's- 1000's ori per chromosome.! #

! • Each ori forms a replication bubble with replication fork at each end. ! • There are multiple enzymes and proteins that are involved in the process.! • DNA replicates in both directions BUT only in the 5'--> 3' direction on the 3' to 5' template strand. ! • DNA polymerase ! ◦ Adds nucleotides ! ◦ Catalyzes formation of phosphodiester bonds ! ◦ Remarkably accurate in proofreading ! ◦ Only makes 1 error in 1 billion of nucleotides. ! # # How do DNA sequences change? --> DNA is continually damaged by chemicals & UV irradiation. # • When exposed to UV irradiation, thymine dimers are formed. ! ◦ UV light adds energy and opens up the double bond in thymine and cytosine, which allows it to react with a neighboring thymine base through covalent bond. ! ◦ A nuclease cuts out a segment of a damaged strand! ‣ The gap is filled by DNA polymerase and DNA ligase. ! # # How does the genome of an organism ever change? • Replication mistakes that cause nucleotide conversion (a DNA polymerase mistake that was not repaired or repaired incorrectly). ! • Mutation (changes in the nucleotide sequence of DNA that are passed on from one cell or organism to another). ! ! # # # # # # #

Mutations: are a fact of life. (Refer to previous page for definition). # • Occur despite proofreading, nucleotide excision, and other repair systems. ! • Mutations can be divided into two types: Somatic mutations, and germ line mutations. ◦ Somatic mutations: occur in somatic (body) cell passed on by mitosis ! ‣ CANNOT be passed on to offspring. ! ‣ Most cancers are the result of DNA damage to somatic cells. ! ‣ There are also inherited cancer alleles that increase the offsprings total lifetime risk! ◦ Germ line mutations: occur in germ cells that give rise to gametes. ! ‣ PASSED on to offspring ! ! Key points regarding translation: • To make proteins during translation, RNA nucleotides will be "read" in codons, which are groups of three nucleotides that identify a specific amino acid. ! • Every amino acid has one or more codon "names"! • The genetic code is a look up table that aligns every possible condon with its amino acid. ! • Reading Frame is the starting point where RNA is "read"! ◦ Since there are 3 nucleotides in a codon, the reading frame will be different if starting at nucleotide1, nucleotide2, or nucleotide 3. ! # ! # ! # # ! # # # • Open Reading Frame will begin with a STOP codon. # ATG is the only codon that starts translation, and end # with a STOP codon (there are 4 of these). # # # Mutations can be classified by their effects on DNA sequence OR the encoded protein: 1. Silent mutations: do not affect gene function. They can be mutations in DNA that is not expressed, or mutations within an expressed region that do not have any effect on the encoded protein. Most mutations in large genomes are silent. Change the DNA sequence but not the amino acid sequence. • Silent mutation often occur in the third position of a codon.

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Missense mutations: result in a change in the coded amino acid, loss of expression of a gene or in the production of a nonfunctional protein or RNA. Usually alter the protein "loss of function". • Missense mutations change the 1st or 2nd position of a codon.

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Nonsense mutation: changes an amino acid to a STOP codon, resulting in premature $ $ termination of translation. #

Frameshift mutation: Deletion or insertion of a number of bases that is NOT a multiple of 3,# which changes the reading frame of RNA, as a result changes the sequence of amino acids# downstream. Usually introduces premature STOP codons in addition to lots of amino acid ! changes.

Conditional mutation: only occurs under certain $ restrictive conditions.# ‣ Wildtype phenotype seen under permissive condition. ! ‣ Many conditional mutants are temperature sensitive ! ‣ Example: cat carries tyrosinase gene that inactive at 37 degree, and active (have color) at 30 degree. ! Chromosomal mutation: MOST dramatic changes $ that occur in genetic material. In other words, # chromosomal mutations is the large scale mutations in the body. Chromosomes can break and# rejoin, which disrupts the sequences of genes. #

What causes mutations to DNA ? • Mutations could be spontaneous or induced by environment. • Spontaneous mutations: are permanent changes in the genetic material that occur without any outside influence. Several mechanisms are involved: ! ◦ DNA polymerase can make errors in replication. ! ◦ The four nucleotide bases of DNA have alternative structures that affect base pairing. ! ◦ Base in DNA may change because of spontaneous chemical reactions. ! ◦ Meiosis is not perfect.! ◦ Gene sequences can be disrupted. ! • Induced mutations: occur when some agent from outside the cell causes a permanent change in the DNA sequence. ! ◦ Chemicals alter the nucleotide bases.! ◦ Some chemicals add groups to the bases ! ◦ Radiation damages the genetic material....