BIO 344 1st Edition Lecture 2Outline of Last Lecture I. Genome Structurea. Comparison of Bacteria and HumanII. Bacterial Genomea. Open reading framesb. Trp operonIII. Human GenomeIV. DNA StructureOutline of Current Lecture I. DNA Topology and TopoisomeraseII. Replicationa. Initiationb. DnaA, DnaB, DnaCc. Polymerases and primersd. Processivity III. Problems with Replicationa. Okazaki Fragmentsb. Catenationc. Completing endsi. telomeresCurrent LectureThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.I. DNA Topology and Topoisomerase- Supercoils are formed to reduce the strain of DNA being unwound during replicationo Supercoils form to compensate for the turn on DNA that has been unwound- Positive supercoiled state (in front of the replication fork in the direction of replication)o =DNA strands are overwound, hinders helix openingo Energy required- Chromosomes in bacteria maintain a negative supercoiled state (behind the replication fork)o Negative supercoil= underwound DNA, facilitates helix openingo Induces torsional strain o Energy is required to do this- Topoisomerase= enzyme that modulates DNA supercoiling to relax the DNAo Gyrase= specialized topoisomerase in bacteria (no histones, unlike humans)o Type I Topoisomerase= cuts one strand of DNA Nucleophile cuts bonds of the backbone to relieve tension Does not require energyo Type II Topoisomerase= cuts two strands of DNA and passes another double strand of DNA through the created path Cuts both strands of DNA, creating a pathway for another two strands of DNA to pass through and creates covalent links to return DNA to relaxed state two supercoils at a timeII. Replication- Initiationo Properties needed for replication origin Negatively supercoiled and A:T richness- Identifier for initiation and binding site In bacteria, oriC= initiation- DnaA= protein that binds to AT repeat sequenceo Binds as a complex to sequence and themselveso AAA + ATPase that binds to oriCo Uses ATP to wrap DNA in positive supercoilso Induces negative supercoils in A:T wich DNA unwinding element, facilitating “melting” of the origin- DnaC= protein that loads helicase onto replication fork- DnaB= replicative helicaseo Helicase= class of enzymes that separates strands, relieves tension of positive supercoilingo Hexomer= 6 subunits of DnaB, with an open center in which DNA strand passes through 6 binding sites for ATP (one on each DnaB monomer) Can bind to ATP (DnaB “grabs” DNA strand) Can also bind to hydrolyzed ATP, ADP (DnaB releases DNA strand) Grabbing and releasing to allow DNA to be pulled through- Leading and lagging strando DNA polymerase and RNA polymerase both replicate in 5’3’ direction and both use 3’ hydroxyl of original strand DNA polymerase requires RNA primer before adding nucleotides- Primase makes RNA primer RNA polymerase can put nucleotides together on its owno Leading strand= continuouso Lagging strand= discontinuous Okazaki fragments= discontinuous DNA fragments of RNA primers and replicated DNA- Processivity= what keeps polymerase associated to the DNA strando Primase has low processivity—adds primer then falls ofo Polymerase has high processivity—stays on Sliding clamp= ring that “follows” polymerase - Clamp stays on until it reaches double stranded DNA and releases DNA polymerase- Prokaryotic clamp is a dimmer, humans have a trimer Clamp loader regulates movement of helicase, sliding clamp, and polymerase to keep everything in sync- Tethering to clamp loader- Protein/protein interaction keeps all initiation and transcription events in proper timing (pictures in slides) Clamp is opened when it binds to ATP- Now having an RNA primer binding site- ATP hydrolyzes to ADP to release the clampo Clamp conformation changes back to closed ringo Single strand binding protein (SSB)= coats single stranded DNA at fork to keep from tanglingIII. Problems with Replication- Okazaki Fragmentso Have pieces of RNA—the RNA primers Need to be replaced with DNAo DNA polymerase uses primers to finish the fragment with DNAo Ligase= links and seals the completed fragments to each other- Cantenation= old and new strand topologically linked togethero Need a Type II Topoisomerase to decantenate- Completing the ends of linear DNAo When last primer is removed from end Okazaki Fragment, DNA polymerase can not add on nucleotides, because there is nothing to start from (see slide picture)o Telomeres= repeating sequences on ends that prevent important sequence from being unreplicated with the shortening of chromosomes Telomerase RNA = template for DNA synthesis that extends the end oforiginal DNA strand so more DNA can be
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