DNA Replication Needed only when a cell is

DNA Replication • Needed only when a cell is about to divide • S phase of cell cycle prior to mitosis or meiosis NO OTHER TIME!!!

Semi-Conservative Replication • Thought most likely mode • contrast w/ conservative and dispersive • Meselson and Stahl • density-gradient centrifugation Cesium chloride

Meselson and Stahl • Produced E. coli with “heavy” DNA • nitrogens were 15 N (extra neutron) • Allowed E. coli to replicate using normal 14 N • Examined how chromosomes separated in gradient

15 N alone 14 N alone semi cons. x x cons. dispers.

15 N alone 14 N alone semi-conservative x x dispersive

15 N alone 14 N alone 1 st replication 2 nd replication

DNA Replication • Bi-directional • must add to 3’ end • Discontinuous • Okazaki fragments • (Multiple origins of replication - euk. ) • Enzyme catalyzed

• Phosphodiester bond “nicked” • nuclease • Helix unwinds • helicase • (topoisomerase) • torsion stress causes H bonds between bases to break • 2 strands separate • single strand binding proteins

• RNA primer • need free 3’ end • RNA primase • removed later • DNA polymerase III • catalyzes phosphodiester bonds between new nucleotides • proofreads • DNA polymerase I • removes/replaces RNA primers • DNA ligase • seals Okazaki fragments

5’ T A LAGGING STRAND G G helicase C Okazaki fragment A 5’ A A RNA primer G G C A C 5’ U T A 3’ C A G C G A T C G G A C A A C T A G C C T G T T DNA polymerase LEADING STRAND

5’ 3’ 5’ lagging strand discontinuous replication Okazaki fragments 5’ 3’ leading strand continuous replication

5’ Polymerase I; ligase 3’ 5’ Okazaki fragments 5’ 3’

Leading vs. lagging. . . lagging leading 3’ 5’ 5’ 3’ leading lagging

Two identical chromatids

Replication • One DNA molecule replicates to form two identical molecules • One chromosome two identical chromatids