18 and 20 September, 2006 Chapter 8 DNA Replication

Overview DNA synthesis requires, primer, template, and dNTPs, and proceeds in the 5’ to 3’ direction on the new strand. The DNA polymerase active site is specific for correctly paired, deoxy nucleotides. DNA polymerase catalysis involves base pairing, and stabilization of the transition state by divalent cations. DNA polymerase is processive, and has a 3’ exonuclease proofreading activity. Synthesis proceeds simultaneously on the leading and lagging strands. DNA synthesis requires Helicase, Primase, SSB, and Topoisomerase. Specialized DNA polymerases have specialized roles in the cell. Sliding clamps increase processivity. Pol III holoenzyme is composed of two pol III cores, a clamp and a clamp loader. The replisome is a complex group of protein activities. DNA replication initiates at origin of replication sequences. DNA replication is highly regulated. Replication of the ends of linear DNA molecules requires special solutions.

DNA Polymerase Substrates

DNA Polymerase Reaction

Base pairing in the DNA Polymerase Active Site

Selection of DNA Monomers

DNA Polymerase Structure Includes two Divalent Cation Cofactors.

Stabilizing the Transition State

Processive Polymerase and Exonuclease Activity

Discontinuous Replication

Primer Replacement

Helicase

SSB

Topoisomerase

Primer Synthesis and Polymerase Exchange

Sliding Clamp

DNA polymerases

The sliding clamp promotes processivity.

Core and Clamp Loading Subunits

Coordination of Leading and Lagging Strand Synthesis

The animation really is worth more that one thousand words.

Origin of Replication

Assembly of replication complexes

DNA must be replicated before anaphase..

Regulation of replication.

Replicating the Ends of Linear DNA

Assay for helicase activity and polarity

ATP control of the clamp loader

Cloning a Replication Origin

Electrophoretic Analysis of Replication.

Methylation

Multiple Rounds of Replication