Lecture 24 - DNA replication Flashcards
Concept of molecular labeling explanation
Introduce labeled precursors (monomers for macromolecules) for a specific period of time called the pulse ->molecules synthesized during the pulse will be labelled
2 ways of labelling molecular precursors
1) Atoms substituted by detectable isotopic variant
2) Chemical derivatives with detectable structural modifications
What experiment determined if DNA replication was conservative or semi-conservative
Meselston-Stahl experiment
What Meselston-Stahl experiment uses
STABLE isotobe 15N, which behaves like normal 14N nitrogen isotope
Normal DNA density
1.7 g/cc
What technique allows separation of 14N and 15N DNA + what medium used
Equilibrium density gradient centrifugation. Uses cesium chloride
First step of Meselston-Stahl exp.
grow E.coli in medium with 15N ammonium salts. All N-containing macromolecules, including DNA, are density labeled w/ 15N
Second step of Meselston-Stahl exp.
wash out 15N medium and grow cells in standard 14 N -> new DNA has normal density
Third step of Meselston-Stahl exp.
Separate old (heavy) and new (light) DNA by equilibrium density gradient centrifugation
DNA replication conservative or semiconservative mechanism
semiconservative
How DNA replication started
Helicase uses energy from ATP hydrolysis to unwind DNA at an A-T rich replication origin
What and in what direction can DNA polymerase extend
Must extend primer, can’t start new chain. Extends 3’ end
Other property of DNA polymerase
Checks previous base-pair before adding next one (3’-exonuclease activity)
Name of protein that puts primer on strand and what it is
Primase. Is an RNA polymerase
Name of DNAPs that extend the primer
First DNA alpha, then DNA delta on lagging-strand mech. or DNA epsilon on leading strand mech.
