DNA Replication Part 2 Enzymology

Figure The Polymerization Reaction

Main E. coli DNA Polymerases DNA polymerase I –Single subunit enzyme –Degrades RNA primers and fills gaps –DNA repair –5'  3' polymerase activity –3’  5’ exonuclease activity – proofreading function –5’  3’ exonuclease activity – primer removal & repair function DNA polymerase III –Main DNA polymerase for replication of genome and plasmids –Multiple subunit holoenzyme – different subunits for pol and exo activities –5'  3' polymerase activity –3’  5’ exonuclease activity – proofreading function

Eukaryotic DNA Polymerases

Catalytic domain of E. coli DNA Polymerase I

holoenzyme Pol III* E. coli DNA Pol III Holoenzyme

Sliding Clamps E. Coli dimer clampHuman trimer clamp Subunit  PCNA

Enzymology of DNA Synthesis DNA-dependent DNA Polymerases –require a DNA template Require a free 3'-OH –a primer NTP added to 3'-OH of growing polynucleotide chain –5'  3' synthesis

Mechanics of DNA Synthesis

Architecture of the Replisome Helicase Topoisomerase ssDNA-binding proteins DNA polymerase RNA primer Leading strand Lagging strand RNA primer Primase

DNA Pol I Finishes the Job DNA Pol I has 5'  3' exonuclease function that destroys RNA primer As RNA is removed, DNA is replaced by the 5'  3' polymerase function DNA ligase seals the gap by joining the 3'OH to the 5'PO 4

Termination of Replication: Circular Chromosomes

Figure What happens at the end of a linear chromosome? The need for a primer poses a problem at the 3’ end of linear chromosomes

Linear eukaryotic chromosomes have telomeres at both ends The term telomere refers to the complex of telomeric DNA sequences and bound proteins Telomeres and DNA Replication

Figure Telomeric sequences consist of hexanucleotide or heptanucleotide repeats The very end of telomeres are a nucleotides 3’ overhang formed by removal of the RNA primer after DNA synthesis Telomere Sequence & Structure

Replication of Telomere Sequences In mammalian somatic cells, telomere repeats are lost with each round of replication Telomere sequence must be maintained in gamete precursors –Done by telomerase Telomerase –utilized a RNA template to extent the 3’ end of one strand at the telomere –telomerase has reverse transcriptase enzyme activity –The RNA is complementary to the DNA sequence found in the telomeric repeat

Figure Step 1 = Binding Step 3 = Translocation The complementary strand is made by primase, DNA polymerase and ligase RNA primer Step 2 = Polymerization Reverse transcriptase Telomerase & Telomere Extension

Telomeres Protect Ends of Chromosomes