BMB 251 1st Edition Lecture 14 Outline of Last Lecture I. GenomeII. Cell divisionIII. DNA conservationIV. ReplicationV. HistonesVI. MutationsVII. DNA “proofreaders”Outline of Current Lecture VIII. DNA replicationa. Leading Strandb. Lagging StrandIX. DNA primaseX. RNA primersXI. RNAse HXII. DNA ligaseXIII. DNA helicase XIV. DNA polymeraseXV. SSB proteinsXVI. Sliding clampXVII. DNA topoisomerases Current Lecture- Clicker Question 1: A mutation in DNA primase would have a bigger impact on which arm of replication?o Lagging strand synthesis- Clicker Question 2: Which polymerase molecule processes on its single-stranded template in the same direction as the helicase?o The polymerase on the leading strand- Only replication in 5’ to 3’ direction allows efficient error correctiono If DNA were synthesized 3’ to 5’, incoming dATP nucleotide would give the activating triphosphate needed for covalent linkage mistakes could not be hydrolyzed away because the bare 5’ chain just created would terminate DNA synthesis- Leading strand only needs DNA polymerase once replication fork has been created - Lagging strand needs more than that for DNA synthesiso DNA primase: enzyme that uses ribonucleoside triphosphate to synthesize…These 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.o …RNA primers: about 10 nucleotides long on lagging strand and at intervals of 100-200 nucleotides; needed for DNA polymerase to bind too RNAse H: erases old RNA primer and replaces it with DNA o DNA ligase: joins 3’ end of new DNA fragment to 5’ end of previous one, connecting the Okazaki fragments and completing synthesis for the segment- DNA helicase: proteins that hydrolyze ATP when bound to single strands of DNA change shapeof protein that allows it to do mechanical worko **Propels itself along DNA and rips double helix apart at 1000 nucleotides per second - Single-strand DNA binding (SSB) proteins (aka helix destabilizing proteins): bind tightly and cooperatively to exposed single-stranded DNA without converting baseso **Bases remain available for templatingo Aid helicase by stabilizing the unwound conformation (cannot unwind helix directly)o **Prevent hairpin helices on single-stranded DNA- DNA polymerase would synthesize a short segment of nucleotides and then fall off if working on its own (quick disassociation helps with Okazaki fragments on lagging strand)o Needs accessory protein call sliding clamp: keeps polymerase firmly on DNA when it is moving but removes it when it runs into double-stranded DNA- Clamp loader: special protein complex that hydrolyzes ATP as it loads the clamp on to a primer-template junctiono DNA polymerase in lagging strand also uses clamp, but releases itself from clamp at end of Okazaki fragment polymerase associates with new clamp on RNA primer of the next Okazaki fragment - Strand-directed mismatch repair: detects potential for distortion in DNA helix form misfit between noncomplementary baseso Is able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand where the replication error occurredo In bacteria: depends on methylation of selected A residues in DNA o In eukaryotes: methylation is not needed to distinguish new from old strand (because many do not methylate their DNA) use marks or “nicks” in between Okazaki fragments to distinguish This means that the leading strand would have to have nicks too, but biologists are uncertain about how this happens- DNA topoisomerases: reversible nuclease that adds itself covalently to DNA backbone PO4 breaks phosphodiester bond in a DNA strando This reaction is reversible, where phosphodiester bond reforms as protein leaveso Topoisomerase (I): produces transient single-strand break (nick) in phosphodiester backbone allows two sections of DNA helix on either side of the nick to rotate freely, using phosphodiester bond in strand opposite of nick as a swivel pointo Topoisomerase (II): forms covalent linkage to both strands of DNA helix at the same time, making transient double-strand break in the helixo **Prevents severe DNA tangling problems that would arise in DNA replication- There are more protein components in eukaryotes replication machinery than in bacterial analogs, but their basic functions are the
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