BMB 462 Lecture 22 Outline of Last Lecture I. Mutations in DNAII. Beneficial Methylation- Begin Unit on Chromosome Structure -III. Composition of the GenomeIV. Supercoiling in ChromosomesV. Function of TopoisomerasesVI. Nucleosome Formation and other Chromosome StructuresVII. Maintenance of Chromosomal StructureOutline of Current Lecture I. Models for DNA ReplicationII. Properties of DNA ReplicationIII. Requirements for in vitro synthesisIV. Comparing the DNA polymerasesCurrent LectureConcepts to remembers from previous courses/lectures:-I. Models for DNA Replicationa. Semi-conservativei. The Watson-Crick modelii. Each daughter chromatid has one original strand of DNA and one new strand of DNA.b. Conservativei. The DNA is used as a template to make a new copy, but then the original strands pair up again and the new strands pair to make an entirely new helix.c. DispersiveThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.i. Each strand is composed of bits and pieces of new DNA and old DNA that is combined together.II. Properties of DNA Replicationa. The Meselson-Stahl experiment: determining that DNA replication is semi-conservative.i. To determine the model for replication, bacteria were grown on 15N media. When the N isotope was incorporated into the nucleotides, all the DNA strands were heavy and appeared at the bottom of the centrifuge tube.ii. In the second generation, they switched to the normal 14N media. The DNA appeared in the center of the tube, showing hybrid helices; heavy and normal isotopes were incorporated.iii. In the next generation, there were light DNA and hybrid DNAb. Bidirectional Replication – Visualized by Cairn via DNA with 3H-labeled thymidinei. When the new DNA nucleotides were labeled with heavy isotopes, 2 areas of heavy-labeled DNA were apparent. The rest of the chromosome had light DNA.1. This indicates that there are 2 replication forks, so replication is bidirectional.c. Origin of Replication – Inman’s denaturation mappingi. Replication doesn't start randomly in the DNA. There are unique sequences that replication originates from.ii. They were able to landmark AT rich regions and then would isolate replicating DNA from a phage. This showed that replication starts at a unique origin.d. DNA replication progresses 5’ to 3’i. DNA is read from 5’ to 3’; new bases are always added to the 3' end of the new strand. 1. This causes problems with the lagging strand, because it runs in the opposite direction.ii. In the leading strand, the strand is read in the correct direction so new bases can be added continuously.e. Semi-discontinuous Replication – Okazaki Fragmentsi. In the experiment, the bases were labeled with 3H thymidine and then the contents were centrifuged. Researchers were able to determine the lagging strand replicated in fragments instead of continuously.III. Minimum Requirements for in vitro synthesisa. Nucleotides – the building blocks. They provide energy as well.b. DNA polymerasec. DNA primers - the primers provide the 3' OH so that the replicating DNA has a base to build off of and determine where replication starts.d. A DNA template (a strand of DNA that gets heated to denature. Then cool it so the primers can anneal so that new bases can be added)e. Buffer and Mg2+IV. Comparing the DNA polymerasesa. General Propertiesi. Polymerases involved in replication are DNA-dependent DNA polymerases- they require a DNA template.ii. All polymerases undergo 5' to 3' synthesis so they require an attached primer with 3' OH1. When the new dNTP is added, the α-phosphate attacks the 3' OH and a new bond is formed.iii. Processive - they can add more than 1 nucleotide before they fall off the DNA (some can add >500,000 nucleotides)iv. Accurate – Polymerases are highly accurate at synthesizing DNA.1. i.e. DNA polymerase III makes 1 error in 1x104 to 105 nucleotides added. DNA polymerase III also has proofreading abilities that increase accuracy to about 1 error in 106-107v. Mismatch repair further increases accuracy to 109-1010vi. The E. coli genome (which is 4.6 x106 base pairs) experiences 1 error every 100 replications. 1. With mismatch repair, that decreases to 1 every 1000 to 10,000 replicationsvii. E. coli adds 1000nucleotides/sec. Eukaryotes add bases slower, approximately 500nucleotides/sec.b. Mechanism for DNA synthesis with Polymerasesi. The primer is complementary to the template strand, which is how the polymerase knows where to attach and start replication. 1. The new nucleotide also has to be complementary to the templatestrand.ii. The phosphate groups are coordinated to Mg2+ in the active site of the polymerase.iii. The incoming dNTP is attacked at the α-phosphate by the 3' OH of the growing DNA chain1. The phospho-anhydride bond is broken and pyrophosphate leaves.iv. A new phosphodiester bond is created.c. Base Selectioni. DNA polymerase I has 2 active sites - one that selects the new nucleotide and one that has proofreading abilities (in the exonuclease active site).1. Replication has to be very accurate so the cell (and more specifically, the polymerase) must take extra precautions to be sure the correct base is added.ii. The exonuclease active site recognizes the shape and size of the correct base pair1. In an incorrect pairing, the exonuclease breaks the bond and expels the incorrect nucleotide so that the correct one can be added in.iii. When incorrect pairing occurs, base pair stacking is impaired because the strand becomes less stable.iv. With base selection alone, there is 1 error every 104 to 105 nucleotides added.d. DNA Proofreading – 3’ to 5’ Exonuclease Activityi. First, a bond is created in the polymerase active site. 1. In incorrect pairing, the base pair gets a weird shape. The polymerase recognizes this and then moves the base pair to the exonuclease active site where the bond is broken.ii. Recognizing the mismatch - the polymerase slides back so that the nucleotide is in the exonuclease site. The phosphodiester bond is broken. 1. It's not a real reversal of the polymerase reaction because that incorrect nucleotide is losta. A dNMP is removed, instead of a dNTP (therefor energy is lost in this process, but it is more crucial that the DNA sequence is accurately preserved)e. Comparing DNA polymerases I, II, and III (E. coli)i. Polymerase III is the major replicative polymerase. ii. Polymerase I
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