MBG 2040 Notes Part 2 Chapter 10: Replication of DNA and Chromosomes Basic Features of DNA Replication In Vivo Importance of DNA replication - Replication ensures that an exact copy of the species’ genetic information is passed from cell to cell during growth and from generation to generation - If DNA failed to replicate itself, the process of mitosis and meiosis would come to a halt - Thus, DNA replication is essential for the continuation of life Standard B-form DNA helix of Watson and Crick - Complimentary base pairs: A with T, G with C - A and G – purines - T, C and U – pyrimidines - Purines = pyrimidines - A + T = G + C - G:C = 3 hydrogen bonds, A:T = 2 hydrogen bonds - Anti-parallel strands – POLARITY - Right handed double helix (B-DNA): most common form in living cells - Ten base pairs per turn - 0.34 nm between stacked bases, 3.4 nm per helical turn Basic features of DNA replication In Vivo (in living) - double helical DNA model predicted replication by a semi-conservative mechanism o 2 copies that each contained one of the original strands and one new strand
- Meselson and Stahl determined semiconservative was the correct copying mechanism for DNA - Discovered this using a technique called cesium chloride equilibrium density gradient centrifugation to separate DNA molecules of different densities - Permits separation of double helical DNA based on its density - Heavier DNA sediments farther down the CsCl gradient - Lighter DNA migrates near the top
Origins - DNA replication is bidirectional from a replication origin o Two replication forks begin at origin and progress bidirectionally around chromosome - Prokaryotes have one origin, which controls replication of a unit of DNA called a replicon - Origin of replication in E. coli is called oriC o 245 nucleotide pairs long, 2 different conserved repeat sequences o one is 13 bp in three tandem repeats rich in A:T, which come apart easily This site of denaturation is essential first step in replication o Second is 9 bp in four repeats interspersed with other sequences These are binding sites for a DnaA that forms replication bubble DNA replication in prokaryotes - DNA polymerase requires free 3-OH on primer strand
o Catalyzes the synthesis of DNA o Cannot begin synthesis without primers (de novo) - Primer DNA with free 3’-OH - Template DNA to specify the sequence of the new strand - Substrates: dNTPs containing bases a, g, c, t - Magnesium - DNA dependent DNA polymerase - DNA synthesis always proceeds in 5’ to 3’ direction Initiation of DNA Replication - Replication of E. coli begins at oriC, with formation of localized region of separation called the replication bubble o Formed by interaction of prepriming proteins with oriC - First step of prepriming is the binding of four molecules of dnaA gene product ( DnaA protein – activates initiation of replication) to the four 9 bp repeats in oriC - Then DnaA proteins bind to form a complex with oriC wrapped around surface o Strand separation begins with three 13 bp repeats and spreads until replication bubble forms -
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