DNA Replication

1. DNA Replication • DNA replication is semiconservative • DNA replication in E. coli • DNA replication in eukaryotes

2. 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

4. 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

5. 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 Viciafaba(broad bean) in 1957 • Experiments with other organisms support semiconservative replication as the universal mode for DNA replication

7. 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

8. 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

10. 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)

11. 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)

12. 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 . . .)

14. 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

15. 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

16. 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

17. 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

19. 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.

20. 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

22. 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

24. http://www.mcb.harvard.edu/Losick/images/TromboneFinald.swf

26. C. Eukaryotic DNA Replication • Eukaryotic chromosomes have multiple origins of replication on each chromosome • There are 6 different eukaryotic DNA polymerases a, d, and e are essential for replication b and z are involved in repair gis only active in mitochondrial DNA replication

27. 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

28. 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

30. How do genes work? RR= • 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?

31. Garrod • Provided the first clue to gene function • studied alkaptonuria, a disease in which homogentisic acid is secreted in the urine. • Hypothesized that the metabolicpathway 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.

33. Garrod

34. 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 Neurosporacrassa • Irradiated mold to create mutants • Tested if they could grow on minimal media • Adrian Srb and Norman Horowitz tested for arginine

35. Beadle and Tatum proposed: “One Gene-One Enzyme Hypothesis” However, it quickly became apparent that… 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). Many biochemical pathways are branched. “One Gene-One Enzyme Hypothesis” “One Gene-One Polypeptide Hypothesis”