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Unformatted text preview: Lecture 16: DNA Replication Semiconservative mechanism- experiment: E. coli cells initially grown in 15N, then transferred into medium of 14N; through density-gradient centrifugation, pattern suggests semiconservative model DNA Polymerases Require a Primer to Initiate Replication- DNA synthesis, like RNA synthesis, proceeds 5' 3' (formation of phosphoester bond between 3' oxygen of growing strand & alpha phosphate of dNTP- primer (preexisting RNA or DNA strand) required to begin chain growth Duplex DNA is unwound + Daughter strands are formed at DNA Replication fork- At replication origins ORI: usually A-T rich; replication fork- helicase unwinds parental DNA strands- primase, a specialized RNA polymerase, forms a short RNA primer complementary to unwound template strands- DNA pol then elongates primer, forming new daughter strand- topoisomerase I: relieves torsional stress produced by local unwinding of duplex DNA- leading strand: 5' 3', can proceed continuously from single RNA primer, in same direction as movement of replication fork- lagging strand: cell synthesizes a new primer every few hundred bases; primers elongated in 5' 3' direction, forming Okazaki fragments; RNA primer of each Okazaki fragment is removed & replaced by DNA chain growth from neighboring Okazaki fragment; ligase joins adjacent fragments; primers are added randomly (not at specific site); o after NDA synthesis begins at primer, primer is removed by RNAse (this is why RNA is used as primer, to recognize easily amidst DNA) o when next Okasaki fragment reaches primer 3' to it, DNA pol adds in nulceotides that were removed by RNAse Several proteins participate in DNA Replication- Replication fork protein A (RPA): ssDNA binding protein that helps stabilize unwound DNA so that it doesn't form secondary structures that would prevent DNA pol from binding to it; dislodged by Pol alpha and pol delta- Proliferating cell nuclear antigen (PCNA): stops synthesis of primer by displacing primase + polymerase alpha (synthesizes primer); keeps DNA polymerase delta (elongates daughter strand) complex connected to DNA; falls off when it hits a primer, along with DNA polymerase DNA Replication occurs Bidirectionally from each origin- experiment: SV40 DNA's replication bubble shows bidirectional replication; edges of bubble are symmetrical to ORI and advance in either direction- two replication forks assemble at a single origin, then move in opposite directions; thus each DNA strand is leading & lagging Origins of Replication- in bacteria: one circular DNA chromosome, one single origin of replication- in eukaryote: multiple linear chromosomes, multiple ORI = solution to replicate such a long DNA in a reasonable time Lecture 17: Chromatin Structure- chromosome chromatin nucleosome octamer of histones- histone: most abundant proteins in chromatin\ Structure of Nucleosomes- DNA component of nucleosomes is much less susceptible to nuclease digestion than is the linker DNA between them...
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