Essential Elements of Biochemistry BCHM 3050 Dr Srikripa Chandrasekaran Lecture 2 20 15 Notes DNA Replication I II III directional nucleotide alike brand new General aspects about DNA replication A Replication starts at a point and then going in both directions bi B Semi conservative C Semi discontinuous one of the strands adds continuously while the other strands adds discontinuously DNA Replication occurs in the 5 to 3 direction A A new nucleotide can only be added to the 3 end of an existing B When a new nucleotide is added the 2 phosphate groups on the end of the existing nucleotide are removed and used for energy Meselson and Stahl Semi Conservative A Meselson and Stahl proved that DNA is semi conservative B Conservative make 2 strands of DNA from scratch that are exactly C Semi Conservative 50 of the new DNA is conserved while 50 is D The first thing done in DNA replication is to break the bonds E Not safe to label phosphorous because it is highly radioactive F Hershey and Chase could not label nitrogen because nitrogen is present in DNA and proteins in the peptide bonds so they would not have been able to differentiate between DNA and proteins so they labeled phosphorus because phosphorus is naturally present in DNA but not in proteins G Meselson and Stahl labeled both strands of DNA with a heavy form of nitrogen one strand labeled N14 and one stand labeled N15 every 2 minutes the DNA splits into 2 1 After 30 minutes they see where the N15 ended up 2 The saw that 50 was the same as the original and 50 was new 3 The detected this by centrifuging it N15 is a heavier isotope form than N14 so N15 sunk to the bottom during centrifuging and N14 floated to the top 4 Only hybrids in generation 1 5 50 hybrids and 50 N14 in generation 2 IV Replication Origins A It is semi conservative break open the strands and new strands are formed B The cell knows to start replication at the origins of replication C The origin has a certain code sequence that signifies that it is a origin of replication 1 V VI D Most eukaryotes have the same or similar sequences at the origins of replication E Replication proceeds in 2 directions replication is always bi F G directional It starts from a spot and branches out 5 to 3 In eukaryotes replication is more complicated because we have many origins of replication spread out through the DNA H The DNA starts breaking at different origins at different spots I Replication bubble refers to the bubble that forms when replication begins at the replication origin and proceeds to replicate out increasing the size of the bubble J All of the strands shown in red are called leading strands these are easier to make because they start at the 5 and goes right along the 3 end K The lagging strands are more difficult to form More general characteristics of DNA replication A Red strand is the leading strand and it pushes the fork open the leading strand is made continuously B The lagging strand which is shown is blue is made discontinuously it is made in pieces at the end all of the pieces are joined together C Together replication is called semi discontinuous since one strand is continuous and one strand is discontinuous D Okazaki who was a Japanese scientist discovered how this replication occurred he discovered that the lagging strand is made in pieces and then the pieces are attached together so the pieces that the lagging strand makes are called Okazaki fragments E The leading and lagging strands operate at the same speed and time they end replication at the same time the lagging strand is NOT slower than the leading strand Summary of DNA replication A The leading strand pushes the replication fork open like a zipper B In order for replication to happen the enzyme that carries out replication DNA polymerase needs a kick start which is the primer C RNA primer serves as a launching pad that allows DNA polymerase to come in a put more nucleotides in to extend the chain D DNA polymerase III the primary enzyme involved in prokaryotic DNA replication it is an enzyme that aids in DNA replication it is a holoenzyme that consists of 17 proteins and contain 2 core polymerases it is the main polymerase in bacteria elongates in DNA replication and has a 3 to 5 exonuclease proofreading ability can add fere nucleotides to only the 3 end of the newly forming strand resulting in elongation of the new strand in 5 to 3 direction can add a nucleotide onto only a preexisting 3 OH group and needs a primer at which it can add the first nucleotide E RNA primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand 2 without an initial RNA or DNA primer for temporary DNA elongation synthesizes a short RNA primer sequence to initiate DNA replication a specialized RNA polymerase that synthesizes short stretches of RNA used as primers F Helicase opens the DNA breaks apart the 2 DNA strands found at the replication fork enzyme that separates double stranded DNA into single strands allowing each strand to be copies G DNA ligase a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond can repair breaks in the DNA by synthesizing a bond between adjoining nucleotides H DNA polymerase I replaces the RNA with the DNA it catalyzes the template directed polymerization of nucleotides into duplex DNA in a 5 to 3 direction possesses a 3 to 5 exonuclease activity or proofreading function which lowers the error rate during DNA replication also contains a 5 to 3 exonuclease activity which enables the enzyme to replace nucleotides in the growing strand of DNA by nick translation 3
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