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Chapter 7

BIOL 205 Chapter 7 Notes.docx

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Queen's University
BIOL 205
Gordon Dueck

BIOL 205 Chapter 7- DNA: Structure and Replication - Griffith used Strep pneumonaie in mice – S strain=lethal, R strain + heat killed S strain is also lethal (first demo that genes are composed of DNA) - Hershey Chase discovered that DNA was the genetic material through T2 phage infecting E. coli and studying the resulting radioactivity - Chargaff – said A=T and C=G and A+G=C+T (purines=pyrimidines) – matched the model (radius of helix) that Watson and Crick later came up with - Rosalind Franklin discovered the double helix via X-ray diffraction - Watson and Crick – 2 antiparallel strands held together by H-bonds between bases of each strand; backbone is alternating phosphate and deoxy sugar connected by phosphodiester bonds - Watson and Crick also referred to semiconservative replication - Meselson and Stahl used heavy 15N and cesium chloride gradient centrifugation to determine the semiconservative nature of DNA replication; they looked at both first and second generation and their results were consistent with the semiconservative hypothesis - DNA polymerases o Poly1 has 3 activities located in different parts of the molecule  Catalyzes chain growth in 5’ to 3’  3’ to 5’ exonuclease activity – removes mismatched bases  5’ to 3’ exonuclease activity – degrade doubles stranded DNA o Poly1 is too slow and too abundant to be responsible for majority of replication o Cairns and DeLucia proved that Poly1 was indeed NOT the polymerase that was mainly responsible for replication when an E. coli strain that had a mutation in the gene that encoded for the poly1 was still able to grow and replicate its DNA o Cairns and DeLucia concluded that poly3 was responsible for catalyzing DNA synthesis at the replication fork - DNA Replication: o Poly 3 (dimer)– adds nucleotides to 3’ growing tip o Leading strand – smooth synthesis o Lagging strand – synthesis is in the “wrong direction”(away from fork)  Okazaki fragments are produced and then ligase binds them o Primer (short chain of nucleotides that binds with the template strand)- needed in order for synthesis of leading strand and okazaki fragments to begin o Primosome – proteins that synthesis primer; major enzyme is primase o Primase – a type of RNA polymerase that synthesizes a short chain of RNA that is complimentary to specific region of chromosome o Poly1- removes RNA primers and fills in remaining gaps with DNA (in between Okazaki fragments) o Ligase- in okazaki fragments it binds 3’ to 5’ end by catalyzing formation of phosphodiester bonds o Good accuracy due to exonuclease activity of poly1 and poly3 that serves as proofreading – excise mismatched bases o Primase lacks proofreading mechanism therefore RNA primer is more likely to have a mutation than DNA - The Replisome: o In E.coli there are only 2 replication forks – each one moves at 1000 nucleotides per second o Polymerase is part of a large nucleoprotein complex = replisome o Pol3 is part of the pol 3 holoenzyme  2 catalytic cores: one for leading, one for lagging strand  accessory proteins bridge the two cores, thus coordinating the synthesis of the leading and lagging strands  Beta clamp (an accessory protein) – keeps pol3 attached to DNA; making pol3 a processive enzyme  Primase is not associated with the clamp protein – it is a distributive enzyme that only adds a few ribonucleotides at a time - Unwinding the double helix: o The replisome contains helicases and topoisomerases o Helicase: disru
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