BIOL 2111 Study Guide - Midterm Guide: Mendelian Inheritance, Dna Polymerase Iii Holoenzyme, Deoxyribonuclease
23 Apr 2016
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Lecture 8: kornberg and others worked out the biochemical aspects of replication in e. coli. Energy for dna synthesis comes from high-energy phosphate bonds associated with dntps. Initiation: proteins open up the double helix and prepare it for complementary base pairing. Elongation: proteins connects the correct sequence of nucleotides on newly formed dna strands. Worked with e. coli cells; goal was to study dna replication in vitro. Step one: develop an in vitro assey for dna synthesis. Grind up cells to botain a cell extract that contains all cellular proteins (including the enzyme that makes dna) Add 14c-thymine deoxynucleotide (14c-dttp) + all dntps + dna. Small amount of 14c-dttp goes from percholoric acid soluble to insoluble (incorporated into dna?) Add dnase enzyme: 14c-dttp goes back into soluble form. Something in the crude cell extract is synthesizing dna. Step two: purify the dna synthesizing activity" from the crude protein fraction.
