DNA Metabolism –DNA replication –DNA repair –DNA recombination Key topics:

What is DNA Metabolism? While functioning as a stable storage of genetic information, the structure of DNA is far from static: –A new copy of DNA is synthesized with high fidelity before each cell division –Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made –Segments of DNA are rearranged either within a chromosome or between two DNA molecules giving offspring a novel DNA DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

Isotope incorporation in 1 round of DNA synthesis The Meselson-Stahl experiment was about the origin of the two strands in each of the daughter genomes Cells were grown on a medium containing only 15 N isotope until all their DNA became fully 15 N labeled Cells were then switched to 14 N medium and allowed to divide once CsCl density gradient centrifugation was used to determine the mass of genomic DNA before and after each round of replication

DNA Replication is Semiconservative The Meselson-Stahl experiment showed that the nitrogen used for the synthesis of new dsDNA becomes equally divided between the two daughter genomes This suggests a semiconservative replication mechanism

Replication of Circular DNA is Bidirectional Both strands are replicated simultaneously

Three DNA Polymerases in E. coli Polymerase I is most abundant but its primary function is in clean-up during replication, repair, and recombination Polymerase II is probably responsible for DNA repair Polymerase III is responsible for DNA replication

Proofreading by 3’  5’ Exonuclease Activity (1) Inherent fidelity of DNA polymerase ~ 1/10 6 Observed Fidelity of Replication ~ 1/10 9

Errors During the Synthesis are Corrected by 3’  5’ Exonuclease Activity (2)

DNA Replication Initiation –At origins (cis acting sites) –Requires initiator proteins (trans-acting factors) –Once per “cell-cycle” –Bidirectional Elongation –Leading and Lagging strands (repeated priming) Termination –Circular and linear chromosomes have unique problems

DNA Replication – Initiation in E. coli

Accesssory proteins DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand Helicases - Open Up the DNA Double Helix in Front of the Replication Fork Single strand binding proteins keep ssDNA out of trouble Clamp subunits tether A Moving DNA Polymerase to the DNA The Proteins at a Replication Fork Cooperate to Form a Replication Machine

DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand

Helicases - Open Up the DNA Double Helix

Single strand binding proteins keep ssDNA out of trouble

The replication machine - expanded

The replication machine -

Other factors operate away from the replication fork A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine DNA Topoisomerases Prevent DNA Tangling During Replication –All topoisomerases form transient covalent phospho- tyrosine bonds to DNA backbone –Type 1 topoisomerase nicks only one strand - unwinds only –Type 2 topoisomerase nicks both strands - unwinds and untangles - Makes DNA “ethereal”

A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Fork

DNA Topoisomerase Prevents DNA Tangling During Replication

Type 1 topoisomerase nicks only one strand - unwinds only

Type 2 topoisomerase nicks both strands - Unwinds and Untangles - Makes DNA “ethereal”

Synthesis of the Leading and Lagging Strands Synthesis always occurs by addition of new nucleotides to the 3’ end The leading strand is made continuously as the replication fork advances The lagging strand is made discontinuously in short pieces (Okazaki fragments) that are then joined

Synthesis of Okazaki Fragments

Coordination of Leading and Lagging Strand Synthesis

DNA Ligase Seals the Nicks in the Lagging Strand

DNA Replication – Termination in E. coli