Chapter 12 DNA Replication and Recombination I. DNA replication Three possible modes of replication A. Conservative entire original molecule maintained B. Semiconservative one strand is template for new one C. Dispersive strands are a mixture of old and new Fig 12.1 Three possible modes of replication I. DNA replication D. Meselson and Stahl (1958) experiment 1. All DNA labeled with 15 N 2. Shift to 14 N for one generation 3. Density gradient centrifugation Fig 12.2 Fig 12.3 Evidence for semi-conservative replication 1
I. DNA replication Three types of DNA replication 1. Theta 2. Rolling circle 3. Linear Theta replication: E. coli and other organisms with circular DNA Fig 12.4 First a brief overview of the differences between these, then the common details. Fig 12.5 Rolling circle replication: some viruses and the F factor Fig 12.6 Linear replication: eukaryotic chromsomes 2
The mantra: DNA synthesis occurs from 5 to 3 on a 3 to 5 template. DNA synthesis occurs by addition of deoxyribonucleotide triphosphates (dntps) Fig 12.7 Fig 12.8 DNA synthesis occurs continuously on the leading strand and discontinuously on the lagging strand Fig 12.1 Fig 12.10 Fig 12.10 Fig 12.10 3
Initiation: Inititator proteins Helicase Single-stranded binding proteins Unwinding: Helicase: binds to the lagging strand template at each fork and moves in a 5 to 3 direction Gyrase: relieves the strain ahead of the replication fork Fig 12.12 Fig 12.11 Priming (primase) Elongation (DNA polymerase III) Fig 12.13 Fig 12.14 Primer replacement (DNA polymerase I) Backbone repair (ligase) Fidelity of DNA replication i) Nucleotide selection ii) Proofreading iii) Mismatch repair (Chapter 17) Fig 12.16 Replication differs at the end Fig 12.19 4
Summary 1. Replication is always semiconservative The mantra: DNA synthesis occurs from 5 to 3 on a 3 to 5 template. 2. Replication always begins at origins 3. DNA synthesis requires a primer 4. Elongation is always 5 to 3 on a 3 to 5 template 5. New DNA is synthesized from dntps Summary 6. Replication is continuous on the leading strand and discontinuous on the lagging strand 7. New strands are complementary and antiparallel to their template 8. Replication is fast and accurate due to precise nucleotide selection, proofreading, and repair Inititator proteins: bind to origin of replication and cause DNA to unwind Single-stranded binding proteins Helicase: binds to the lagging strand template at each fork and moves in a 5 to 3 direction; break H-bonds Gyrase: (topoisomerase) relieves the strain ahead of the replication fork; dsdna break and resealing Primase: lays down the RNA primer DNA polymerase III: replication of DNA beginning at primer and extending from 5 to 3 DNA polymerase I: primer replacement Ligase: backbone closure Eukaryotic DNA polymerase The end replication problem There are at least 13! (Table 12.5) DNA pol α: primase activity DNA pol δ: replication on leading and lagging strand DNA pol β: recombination and repair DNA pol γ: mtdna replication Fig 12.19 5
Fig 12.20 ssdna end is G-rich repeat RNA of telomerase is complementary Telomerase synthesizes complementary DNA RNA template moves Etc II. Recombination A. Involves breakage/reunion of both strands Synthesis on complementary strand Fig 12.22 Fig 12.22 If crossing over took place before DNA synthesis it would look like this: but crossing over results in recombinant and nonrecombinant products. Therefore it must take place after DNA synthesis 6
V. Recombination B. Holliday model: single stranded break model Fig 12.23 1. Break both strands of each duplex Note spelling: Holliday, not holiday. Fig 12.23 2. Broken strands pair with complement in other duplex Fig 12.23 4. Strand migration 3. Ligation Fig 12.23 Fig 12.23 4. Resolution of the complex 7
Fig 12.23 Fig 12.23 Two possible types of resolution: Fig 12.23 C. Double-stranded break model 1. Double strand break in one chromosome 2. Removal of nucleotides on one strand 3. 3 end displaces strand on unbroken chromosome 4. DNA synthesis from the 3 end C. Double-stranded break model 5. Displaces strand pairs with broken end 6. DNA synthesis from the 3 end of nonmigrating strand 7. Ligation to form two Holliday junctions 8. Resolution of the junctions 8
D. Enzymes involved in recombination 1. RecA single strand invasion 2. RecBCD unwinds double-stranded DNA 3. RuvA, RuvB branch migration 4. Resolvase (RuvC) cleaves Holliday structures 5. Single-stranded binding proteins 6. Ligase 7. DNA polymerases 8. gyrase 9