DNA REPLICATION • Complementary • Semiconservative • Bidirectional • Occurs in the S phase • Synthesis in the 5' to 3' direction – antiparallelKEY ENZYMES • Helicase separates DNA strands at replication fork • Topoisomerase relieves supercoiling downstream of replication fork • Primase synthesizes RNA primer • DNA polymerase synthesizes DNA • Ligase joins DNA fragments togetherReplisome complex • Protein complex assembled at each replication fork • Comprises: helicase, primase, DNA polymerases, single-strand binding proteinsKey differences between Prokaryotes and Eukaryotes • Eukaryotes have multiple origins of replication • Different DNA polymerases, helicases, topoisomerase and ligase • Eukaryotes have nucleosomes and telomeres • Eukaryotes typically have more DNAProkaryotic DNA Polymerases • DNA pol III: synthesizes both the leading and lagging strands. • DNA pol I: removes RNA primers and fills the remaining gaps with DNA nucleotides.Eukaryotic DNA polymerases • DNA pol epsilon: elongates leading strand DNA. • DNA pol delta: elongates lagging strand DNA and displaces RNA primers from the lagging strand. • DNA pol alpha: complexes with primase to synthesize primers that comprise RNA and DNA. PROKARYOTIC REPLICATION (E. COLI) • DNA is melted at one origin of replication (region rich in adenine and thymine) • 2 replication forks with replisomes on each side of origin are created (bidirectional replication) • Helicase separates parent DNA strand (breaks H-bonds) • Single-stranded DNA binding proteins stabilize strands (prevent degradation) • Primase synthesizes RNA primers (~ 10 nucleotides long) • DNA polymerase elongates strand (adds to 3' end only) • Leading strand: oriented 3' to 5' towards fork • Lagging strand: oriented 5' to 3' towards fork --> Okazaki fragments (discontinuous replication) • Different DNA polymerase removes primer & fills gap with DNA • DNA ligase joins Okazaki fragments • Topoisomerase relieves supercoiled DNA downstream of replication fork