DNA Replication A. DNA replication is semiconservative B. DNA replication in E. coli C. DNA replication in eukaryotes 1

A.... Semiconservative In DNA replication, the two strands of a helix separate and serve as templates for the synthesis of new strands (nascent strands), so that one helix gives rise to two identical “daughter” helices 2

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A.... Semiconservative Hypothetically, there could be three possible ways that DNA replication occur: –Conservative replication: One daughter helix gets both of the old (template) strands, and the other daughter helix gets both of the new (nascent) strands –Semiconservative: Each daughter helix gets one old strand and one new strand –Dispersive: The daughter helices are mixes of old and new 4

A.... Semiconservative Two major lines of experiment in the mid 1950s – early 1960s demonstrated that DNA replication is semiconservative, both in prokaryotes and eukaryotes: –Meselson and Stahl demonstrated semiconservative replication in Escherichia coli in 1958 –Taylor, Woods, and Hughes demonstrated semiconservative replication in Vicia faba (broad bean) in 1957 –Experiments with other organisms support semiconservative replication as the universal mode for DNA replication 5

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B.Replication in E. coli DNA replication is semiconservative and requires a template Deoxynucleoside triphosphates (dNTPs) (dATP, dTTP, dGTP, dCTP) are the “raw materials” for the addition of nucleotides to the nascent strand 7

B.Replication in E. coli Nucleotides are added only to the 3´ end of a growing nascent chain; therefore, the nascent chain grows only from the 5´  3´ direction The addition of nucleotides to a growing chain is called chain elongation 8

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B.Replication in E. coli Addition of nucleotides to a nascent chain is catalyzed by a class of enzymes called DNA-directed DNA polymerases (or DNA polymerases, for short) E. coli has three DNA polymerases (I, II, and III) 10

B.Replication in E. coli –DNA polymerase I was discovered in the mid 1950s by Arthur Kornberg (it was originally simply called “DNA polymerase” –DNA polymerase I has three different enzymatic activities: 5´  3´ polymerase activity (elongation) 3´ exonuclease activity (proofreading function) 5´ exonuclease activity (primer excision) 11

B.Replication in E. coli –The 3´ exonuclease activity of DNA polymerase I performs a “proofreading” function: it excises mismatched bases at the 3´ end, reducing the frequency of errors (mutations) –The 5´ exonuclease activity is responsible for RNA primer excision (see later...) 12

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B.Replication in E. coli –By the late 1960s, biologists suspected that there must be additional DNA polymerases in E. coli (to account for the rate of replication observed in experiments) –In the early 1970s, DNA polymerases II and III were discovered 14

B.Replication in E. coli –DNA polymerases II and III each have two enzymatic activities: 5´  3´ polymerase activity (elongation) 3´ exonuclease activity (proofreading) –Neither has the 5´ exonuclease activity –DNA polymerase III is the enzyme responsible for most of the nascent strand elongation in E. coli 15

B.Replication in E. coli DNA polymerase can only elongate existing chains; it cannot initiate de novo chain synthesis –Nascent strand initiation requires the formation of a short RNA primer molecule –The RNA primers are synthesized by RNA primase (a type of 5´  3´ RNA polymerase, capable of initiating nascent chain synthesis from a DNA template; uses ribose NTPs as nucleotide source) –The primers are eventually excised by the 5´ exonuclease activity of DNA polymerase I 16

B.Replication in E. coli Replication begins at a location on the chromosome called the origin of replication (ori), and proceeds bidirectionally. As the DNA helix unwinds from the origin, the two old strands become two distinctive templates: –the 3´  5´ template, –and the 5´  3´ template 17

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B.Replication in E. coli –Replication on the 3´  5´ template is continuous (leading strand synthesis), proceeding into the replication fork –Replication on the 5´  3´ template is discontinuous, resulting in the synthesis of short nascent segments (lagging strand or Okazaki fragments), each with its own primer –After primer excision is complete, nascent segments are “sealed” (the final phosphodiester bond is formed) by DNA ligase –DNA polymerase III may be able to synthesize both the leading and lagging strands simultaneously by having the 5´  3´ template to fold back. 19

B.Replication in E. coli Several proteins are required to unwind the helix –Helicases dnaA protein recognizes the origin, binds, and begins the separation of the helix dnaB dissociates from dnaC; the dnaB is responsible for moving along the helix at the replication fork, “unzipping” the helix 20

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B.Replication in E. coli –DNA gyrase Makes temporary single-stranded “nicks” (single PDE bond breaks) in one of the two template strands to relieve the torsional stress and supercoiling caused by the unwinding of the helix –Single-stranded binding proteins (SSBPs) Bind to the unwound strands of the template, stabilizing the single-stranded state long enough for 22

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C.Eukaryotic DNA Replication Eukaryotic chromosomes have multiple origins of replication on each chromosome There are 6 different eukaryotic DNA polymerases  and  are essential for replication  and  are involved in repair  is only active in mitochondrial DNA replication 26

C.Eukaryotic DNA Replication Eukaryotic chromosomes are linear, not circular like prokaryotic chromosomes –The ends of eukaryotic chromosomes are formed by an enzyme called telomerase –Telomerase adds repeats of TTGGGG to the 3´ ends of eukaryotic chromosomes –The repeats fold over into a “hairpin” structure, providing a primer for completion of the end (telomere) structures 27

C.Eukaryotic DNA Replication –In most eukaryotic somatic cells, the telomerase activity stops shortly after the cell differentiates. –After this, the chromsomes gradually shorten with each division –The loss of telomerase activity is a major factor in cell aging 28

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How do genes work? Genes carry the instructions for making and maintaining an individual But how is this information translated into action? How does an organism’s genotype specify its phenotype? RR= 30

Garrod Provided the first clue to gene function studied alkaptonuria, a disease in which homogentisic acid is secreted in the urine. Hypothesized that the metabolic pathway in which homogentisic acid is an intermediate must be blocked in alkaptonurics Block due to lack of an enzyme that breaks down homogentisic acid, leading to its buildup. 31

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Garrod 33

George Beadle and Edward Tatum Developed the one-gene, one-enzyme hypothesis from Garrod’s work –Each gene carries the information for one protein or enzyme. Did experiments using red bread mold Neurospora crassa –Irradiated mold to create mutants –Tested if they could grow on minimal media Adrian Srb and Norman Horowitz tested for arginine 34

Beadle and Tatum proposed: “One Gene-One Enzyme Hypothesis” However, it quickly became apparent that… 1.More than one gene can control each step in a pathway (enzymes can be composed of two or more polypeptide chains, each coded by a separate gene). 2.Many biochemical pathways are branched. “One Gene-One Enzyme Hypothesis” “One Gene-One Polypeptide Hypothesis” 35