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UD BISC 207 - Chapter 12 – DNA Replication

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Chapter 12 – DNA Replication & Manipulation (S-Phase of Cell Cycle)*Happens during Mitosis/Meiosis only; DNA transcription/translation is a different process I. Review of DNA Structure 2 nucleotides are linked by series of covalent bonds called a phosphodiester bond (C-O-P-O-C)A. The Backbone1. Linear strand of alternating phosphate & sugar groups2. Negatively charged 3. Tail-end phosphate (not connected to a sugar) will be HPO4 (Hattached to top O), instead of PO4 (see diagram)4. All sugars in strand are oriented in same direction (5’  3’ or3’  5’)5. Strand has polarity or directionB. Base Sequence1. Variable part of DNA – holds infoC. Double-helix 1. 2 strands stay together by H-bonds that form between complimentary bases (1 purine—1 pyrimidine)a. A-T, G-Ci. This specificity is what keep width of DNA constantb. Bases also interact noncovalently with the ones above & below and “stack” (tightly group), which stabilizes helixII. How DNA Replicates A. Semi-Conservative replication: each new double helix has one daughter strand & one parental strandB. Starts at sites called “origins”C. Bacteria have 1 small, circular chromosome with one origin:III. Replication fork Diagram A. Bidirectional replication1. Replication occurs in both directions from the originB. Each new strand:1. Has the opposite orientation from the parent strand2. Forms in its 5’3’ directiona. DNA polymerase can only move from 5’3’ on daughter strandC. At each origin, the lagging strand is built in one piece; the lagging strand is built in pieces (Fig. 12.5)IV. DNA Replication (Fig. 12.5) A. General1. Leading Strand: Parent strand that is unzipped in its 3’5’ direction2. Lagging Strand: Other parent strand that is unzipped in its 5’3’ direction3. Both daughter strands are synthesized in the 5’3’ directiona. For the leading strand, that doesn’t cause a problemb. For lagging strand, DNA is replicated in fragments4. Happens during S-phase of cell cycleB. Enzymes/Proteins & Functions1. DNA Polymerase – creates (“elongates”) daughter DNAa. Also “proofreads” new DNA to make sure there are no mistakes in it2. DNA Helicase – unzips existing DNA3. RNA Primase – adds a small RNA sequence to unzipped DNA (“primer”)4. Single-Stranded Binding Proteins – prevent unzipped DNA from coming back together5. DNA Topoisomerase – relieves stress on DNA during unzipping6. DNA Ligase – glues together Okazaki fragments on lagging strandC. Process1. DNA Helicase hydrolyzes ATP to unwind the existing double helixa. As it unwinds, the replication bubble formsi. In that bubble, both single strands of existing DNA are replicated by DNA polymerase (see diagram)b. Single-stranded binding proteins prevent separated parent strands from coming back togetherc. DNA topoisomerase relieves the stress of uncoiling ahead of the replication fork so DNA doesn’t break2. In the bubble, DNA polymerase elongates the existing strands, starting at their 5’ ends3. To start adding nucleotides, DNA polymerase needs a starting sequencea. To make DNA polymerase start working, RNA primase makes a short strand of RNA (“primer”)b. DNA polymerase extends the primeri. On the lagging strand, DNA polymerase will run into the primer of the preceding fragment- A different form of DNA polymerase replaces these primers with DNA4. On the lagging strand, DNA ligase anneals Okazaki fragmentsV. Lots of Equipment is involved (fig. 12.8) (Process reiterated) A. Red piece is the starting piece of the light blue strand (starting strand)1. You get this from RNA primasea. Makes a short strand of RNA called a “primer”. DNA polymerase extends the primer (Fig. 12.6)B. On the lagging strand, DNA polymerase will run into the primer of the preceding fragment (Fig. 12.5.3) (Okazaki fragment)a. When that happens, a different DNA polymerase from removes the primer and fills in the DNA (fig. 12.6.3)C. DNA ligase joins the DNA fragments (Fig. 12.6)VI. SummaryA. RNA primase makes primersB. One type of DNA polymerase extends strandC. MAKE ENZYME/FUNCTION LIST FOR THIS CHAPTERD. A 23nd type of DNA poly removes any primers & fills in DNAE. DNA ligase bonds any fragmentsVII. Most types of DNA polymerase can “proofread”A. Checks that the most recently added nucleotide is correct, if not, it will cut it out and add the correct one (fig. 12.7)Replication ForkReplication Fork: where parental strands separate3’5’Formation of daughter strandFormation of daughter strand3’5’Replication Bubble Origin of

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