3 2 2014 CHAPTER 15 DNA and the Gene Synthesis and Repair o Hershey Chase Experiment hypothesized that viral genes consist of DNA or protein strategy based on 1 proteins contain S but not P 2 DNA contains P but not S viruses labeled w radioactive isotope of P or S bacteria infected with viruses cultures agitated to separate empty viral capsids from bacteria cells solutions centrifuged radioactive DNA in pellet radioactive protein in solution CONCLUSION viral genes consist of DNA DNA is the hereditary material o capsid exterior protein coat of the original parental virus o Watson and Crick proposed antiparallel fashion two DNA strands line up in opposite direction to each other double helix double stranded molecule that forms from antiparallel strands twisting around each other bc of complementary base pairing or H bonding particular base pairs A T G C each strand of DNA has a directionality polarity one end has hydroxyl group on 3 C of deoxyribose one end has phosphate group on 5 C CONCLUSION viral genes consist of DNA DNA is the hereditary material existing strands of DNA are template for the production of new strands where bases were added according to complementary base pairing o DNA s primary structure backbone made of sugar phosphate groups of deoxyribonucleotides nitrogen containing bases that project from backbone directionality DNA has a 3 end and 5 end on the 3 carbon of deoxyribose hydroxyl group on the 5 carbon phosphate group o alternative hypotheses for how old new DNA strands interact during replication 1 Semiconservative replication parental DNA strands separate each used as template for synthesis of new strand output daughter molecules have 1 old and 1 new strand 2 Conservative replication 3 Dispersive replication parental molecule is template for synthesis of entirely new molecule parent molecule cut into sections daughter molecules have old DNA interspersed w newly synthesized DNA o Meselon Stahl discovered each parental DNA strand copied entirely failed to illustrate mechanism for process o DNA polymerase enzyme that catalyzes DNA synthesis its discovery enabled understanding of DNA replication reactions can only work in one direction add deoxyribonucleotides to only the 3 end of a growing DNA chain synthesizes DNA in the 5 3 direction DNA polymerization exergonic monomers that act as substrates in rxn are deoxyribonucleoside triphosphates dNTPs dNTPs have high PE bc of 3 phosphate groups the d stands for any of the 4 bases found in DNA A T G C o o replication bubble bubble that forms when DNA is being synthesized forms in a chromosome that is being actively replicated grows as GNA proceeds bc of bidirectional synthesis forms the origin of replication specific sequence of bases 1 ooR in bacteria many in eukaryotes bidirectional replication DNA synthesis occurs and replication bubbles grow in both directions simultaneously 3 2 2014 o o occurs only in eukaryotes bc of multiple origins of replications replication bubbles replication fork y shaped region where parent DNA helix split into 2 separate strands before copying enzymes that open and stabilize DNA double helix enzyme helicase catalyzes breaking of H bonds btwn 2 DNA strands to separate them single strand DNA binding proteins SSBPs attach to separated strands to prevent them from closing enzyme topoisomerase cuts and rejoins DNA downstream of replication fork to relieve tension from unwinding o DNA polymerase requires a primer few nucleotides bonded to template strand that give a 3 hydroxyl group that can combine with incoming dNTP to form a phosphodiester bond primase type of RNA polymerase synthesizes short RNA segment that serves as primer DNA polymerase III adds bases to 3 end of primer produces leading continuous strand synthesized CONTINUOUSLY towards replication fork in 5 3 direction lagging discontinuous strand other DNA strand synthesized DISCONTINUOUSLY away from replication fork occurs bc DNA synthesis must proceed in 5 3 direction synthesis begins when primase synthesizes short RNA segment that serves as primer DNA polymerase III adds bases to 3 end of primer o DNA polymerase moves away from replication fork as helicase continues to open it and expose single stranded DNA on lagging strand o Discontinuous Replication hypothesis along lagging strand once primase synthesizes an RNA primer on lagging strand DNA polymerase might synthesize short fragments of DNA fragments linked together to form whole continuous strand tested by Okazaki and his colleagues Okazaki fragments the lagging strand is synthesized as short discontinuous fragments DNA polymerase I removes RNA primer beginning of each Okazaki fragment to fill in gap DNA ligase enzyme that catalyzes formation of phoshopdiester bond btwn adjacent fragments appears Discontinuous strand name comes bc Okazaki fragments are synthesized independently and joined together later o o replisome telomere o telomerase a large macromolecular machine containing joined enzymes responsible for DNA synthesis around replication fork region at end of linear chromosome that does not contain genes but consists of short repeating stretches of bases replication of telomeres can be problematic telomere on lagging strand shortens during DNA replication replication fork reaches end of linear chromosome no way to replace RNA primer from lagging strand w DNA bc no primer available for DNA synthesis primer removed leaving section of single stranded DNA lagging strand one end of each new chromosome remaining single stranded DNA eventually degraded shortens chromosome enzyme that adds more repeating bases to end of lagging strand with own RNA template catalyzes synthesis of DNA from RNA template carried w it primase makes RNA primer that DNA uses to synthesize lagging strand enzyme ligase connects new sequence and prevents lagging strand from shortening with each replication o DNA replication is very accurate avg error rate 1 per billion bases o DNA polymerase is highly selective 3 2 2014 DNA polymerase only inserts the wrong bases when matching complementary bases 1 100 000 bases added o DNA polymerase proofreads work DNA polymerase checks match between paired bases corrects mismatched bases when they occur If a mismatch is found DNA polymerase removes mismatched base that was just added possible bc epsilon subunit of DNA polymerase III acts as an exonuclease and removes deoxyribonucleotides from DNA reduces error rate to 1e 7 if DNA polymerase fails to correct other
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