1. Chapter 25 DNA Metabolism Replication, Repair and Recombination Semiconservative DNA replication Each strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized strand Meselson-Stahl Experiment 1953 Watson-Crick Structure 1957 This experiment proves the semi-conservative model of DNA replication Nitrogen-15 isotope used (heavy) Nitrogen-14 isotope (most common, light)

2. E. Coli grown on 15 N first Transferred to 14 N medium Samples withdrawn at different generations Subjected to CsCl gradient centrifugation

3. Chromosomal DNA Replication is Bidirectional E. coli chromosome is circular, double-stranded DNA (4.6x10 3 kilobase pairs) Replication begins at a unique site (origin) Proceeds bidirectionally until the two replication complexes meet (termination site) Replisome - protein machinery for replication (one replisome at each of 2 replication forks) Duplication in about 38 minutes Bidirectional DNA replication in E. coli New strands of DNA are synthesized at the two replication forks where replisomes are located

4. Eukaryotic replication Eukaryotic chromosomes are large linear, double- stranded DNA molecules Fruit fly large chromosomes ~5.0x10 4 kb (~10x larger than E. coli) Replication is also bidirectional Multiple sites of initiation of DNA synthesis (versus one site in E. coli) Replicating DNA in the fruit fly Large number of replication forks at opposite ends of “bubbles” of duplicated DNA

5. DNA Polymerase E. coli contains at least 5 DNA polymerases DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand DNA polymerase II - role in DNA repair DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation Chain Elongation Is a Nucleotidyl- Group-Transfer Reaction Base pair between incoming deoxynucleotide 5' triphosphate (blue) and a residue of the parental strand Terminal 3' OH attacks a-phosphorous of incoming nucleotide to form new phosphodiester linkage Strand growth always in the 5' → 3' direction Irreversible due to formation or PP i which is quickly hydrolyzed to 2P i by pyrophosphatase

6. DNA PolIII Remains Bound to the Replication Fork DNA polymerase III is a processive enzyme (remains bound to the replication fork until replication is complete) Certain subunits form a sliding clamp which surrounds the DNA molecule Two  -subunits associate to form a head-to-tail dimer in the shape of a ring that completely surrounds the DNA Remaining subunits of DNA pol III are bound to this structure

7. Bacteriophage DNA polymerase bound to DNA Proofreading Corrects Polymerization Errors Recall enzymes that degrade polynucleotides Exonucleases – degrade from chain ends Endonucleases – cut at recognized interior sites DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity Pol III can catalyze both chain elongation and degradation Recognizes distortion in the DNA caused by incorrectly paired bases Exonuclease activity removes mispaired nucleotide before polymerization continues

8. DNA Polymerase Synthesizes Two Strands Simultaneously DNA pol III catalyzes chain elongation only in the 5' 3'direction (antiparallel DNA strands) Leading strand - synthesized by polymerization in the same direction as fork movement - continuous replication -polynucleotide (from origin to the termination site) Lagging strand - synthesized by polymerization in the opposite direction of fork movement -discontinuous replication in short pieces (Okazaki fragments) - Pieces are joined by a separate reaction Two core complexes of DNA pol III, one for leading, one for lagging strand

9. RNA Primer Begins Each New Strand, including each Okazaki Fragment Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging- strand template) DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis Replisome - includes primosome, DNA pol III Okazaki fragments are joined to produce a continuous strand of DNA in 3 steps: (1) Removal of the RNA primer (pol I) (2) Synthesis of replacement DNA (pol I) (3) Sealing of adjacent DNA fragments (DNA ligase)

10. DNA polymerase I activities The 5‘ 3' activity of DNA pol I removes the RNA primer at the beginning of each Okazaki fragment Synthesizes nick translation: polymerase activity synthesizes DNA in place of RNA Nick – Break in the DNA backbone

11. Klenow (large) fragment of DNA pol I, lacks 5'→3' exonuclease activity Used for DNA synthesis DNA ligase activity Catalyzes the formation of a phosphodiester linkage between 3’-hydroxyl and 5’-phosphate of adjacent Okazaki fragments Eukaryotic enzymes require ATP cosubstrate E. coli DNA ligase uses NAD + as a cosubstrate Model of the Replisome Replisome contains: a primosome, DNA polymerase III holoenzyme, additional proteins DnaB helicase is part of the primosome and facilitates unwinding of the DNA helix Topoisomerases relieve supercoiling ahead of the replicating fork (not part of the replisome) Single-stranded binding proteins (SSBs) stabilize single-stranded DNA

12. 3 Stages of DNA Replication in E. coli 1. Initiation 2. Elongation 3. Termination Initiation Regulated for once per cell cycle Replisome assembles at origin site (oriC) Origin site is a highly conserved sequence and contains two series of short repeats DnaA is first initiation protein binds at four 9 bp repeat sequences causes denaturation at three 13 bp repeats requires ATP and HU (histone-like protein) Hexamers of DnaB (aided by DnaC) unwind DNA

14. Elongation DNA helicases – unwinding ahead of fork SSB’s stabilize single strands Primase – synthesizes RNA primers -1 for leading strand - 1 for each Okazaki fragment

15. Core – catalyzes polymerization reaction  subunits – clamp for processivity Lagging strand -  subunits load template onto  clamp - New one every Okazaki fragment

16. ~1000 nucleotides/s Process completed by DNA pol I and DNA ligase

17. Termination Terminator utilization substance (Tus) binds to the ter site Tus inhibits helicase activity and thus prevents replication forks continuing through this region ter sites act as replication “block” Last few hundred bp made by unknown mechanism Topoisomerase IV frees catenated DNA’s Catenanes - circles wound around each other

18. DNA Replication in Eukaryotes Mechanism similar to that in prokaryotes: leading strand continuous synthesis, lagging strand discontinuous synthesis Replication forks move more slowly, but many replication forks (~50 nucleotides/s) Okazaki fragments are shorter in eukaryotes (~100-200 residues) At least 5 different DNA polymerases

19. Accessory proteins associated with the replication fork PCNA (proliferating cell nuclear antigen) forms structure resembling  -subunit sliding clamp (E. coli DNA polymerase III) RPC (replication factor C) similar to  complex of DNA pol III RPA (replication factor A) similar to prokaryotic SSB Helicases also present to unwind DNA