which experiment was used to find Semiconservative DNA in eukaryotestaylor woods hughestaylor-woods-hughes
- vicia faba was used to demonstrateDNA replication is semiconservative in eukaryotesthe taylor-woods-hughs experiment monitored process of replication with labeled3H-thymidine and performed autoradiography- Pinpoints location of radioisotope in cell
- Photographic emulsion placed over cellular material and stored in the dark
- Develops much like photographic filmautoradiographyresult of autoradiographypresence of dark grains identifies location of newly synthesized DNADNA replication begins at theORIoriorigin of replicationat the site of replication, helix is unwound, creatingreplication forkreplication is ___, meaning there are two replication forksbidirectionalLength of DNA replicationrepliconhow man ORI does bacteria haveoneWhere does DNA synthesis originate in bacteriaoriChow many base pairs is in the e.coli genome4.6 million___ replicon consists of its entire genomee.coliDNA synthesis in bacteria involves ___ polymerasesfivewhat are the five polymerases in DNA synthesis calledDNA Polname the DNA Polymerase:
Isolated enzyme from E.coliiname the DNA Polymerase:
enzyme directs dna synthesisiname the DNA Polymerase:
requires DNA template and all for dNTPsiname the DNA Polymerase:
enzyme consists of polypeptide w/ 928 aminoacidsiwhich direction does chain elongation by DNA polymerase i occur in5 to 3chain elongation by DNA Pol I
- nucleotide added, ______ cleaved off, providing newly exposed 3'OHtwo terminal phosphates3'OH can participate in addition of another ___ as DNA synthesis proceedsnucleotide- can elongate existing DNA strand
- cannot initiate DNA synthesisDNA Pol I, II, IIIproofread newly synthesized DNA, remove/replace incorrect nucleotidesexonuclease activity 3-5only dna polymerase 1
excises primers, fills in gaps left behindexonuclease activity 5-3active form of DNA polymerase IIIholoenzymeholoenzymes contains ...core enzyme complexes made up of subunitsalpha 5'-3' function5-3 polymerizationsigma 3'-5' function3-5 exonucleasetheta functioncore assembly1/7 key issues that must be resolved during DNA replicationunwinding of helix2/7 key issues that must be resolved during DNA replicationreducing increased coiling generated during unwinding3/7 key issues that must be resolved during DNA replicationsynthesis of primer for initiation4/7 key issues that must be resolved during DNA replicationdiscontinuous synthesis of second strand5/7 key issues that must be resolved during DNA replicationremoval of the RNA primers6/7 key issues that must be resolved during DNA replicationjoining of gap-filling DNA to adjacent strand7/7 key issues that must be resolved during DNA replicationproofreadinginitiator protein is encoded bydnaAbinds to ORI causing conformation changedna Acauses helix to destabilize and open up and exposes ssDNAdnaAmade of dnaB polypeptides
hexamer of subunits assembles around exposed ssDNADNA helicaseSubsequently recruits holoenzyme to bind replication fork and initiate replicationdna helicasehelicase require energy supplied byhydrolysis of atpstabilize the open conformation of helix
bind specifically to single strands of dnasingle straanded binding proteinsssbpsingle stranded binding proteinsenzyme relieves coiled tension from unwinding of helix
member of large enzyme group DNA topoisomerasedna gyrasewhat enzyme group is dna gyrase part ofdna topoisomerasemakes single or double stranded cuts
driven by energy released during ATP hydrolysisdna gyraserecruited to replication form by helicase
sythesizes rna primer
provides 3'-OH required by DNA polymerase III for elongationPrimase: RNA polymeraseremoves primer and replaces it with DNADNA polymerase Idna polymerase i
_____ and replaces it with DNAremoves primeruniversal phenomenon
found in bacteria, viruses, and several eukaryotic organismsRNA primingtwo strands of double helix are ____antiparallelDNA polymerase III only synthesizes5-3Continuous DNA synthesisleading stranddiscontinuous dna synthesislagging strandlagging strand synthesized as ___, each with rna primerokazaki fragmentsremoves primers on lagging stranddna polymerase icatalyzes formation of phosphodiester bonds
seals nicks and joins fragmentsdna ligaseboth dna strands synthesized ___concurrentlylagging strand is __looped___ prevents core enzyme dissociation from templatedna clampintegral part of DNA replication
synthesis of noncomplementary base pairs inserted occasionally
dna polymerase exonuclease activity of 3-5 allows for excise of nucleotidesproofreading and error correcting___ are essential to all DNA synthesisenzymes and proteinsinterrupt or impair aspects of replicationmutationsexample of mutationlethal mutationsligase deficient mutation and proofreading deficient mutations are examples oflethal mutationsexpressed under specific conditionsconditional mutationsexample of conditional mutation
may not be expressed at particular permissive temperature
mutant cells grown at restrictive temperature and mutant phenotype expressedtemperature-sensitive mutationeukaryotic and bacterial dna replication shares many featuresdouble stranded DNA unwound at ori
replication forks formed
bidirectional synthesis creates leading and lagging strands
eukaryotic polymerases require 3 dna-triphosphates, template and primer___ chromosomes contain multiple ORIseukaryotic chromosomesyeast genomes contain ___ origins250-400ARSsautonomously replicating sequencessequence that is the same in all yeast ARSsconsensus sequenceyeast ori has ___ base pairs of consensus sequences120pre-rcpre-replication complexassembles at replication ORIspre-rcorigin recognizing complex (ORC) recognizesoriDNA polymerases are involved innuclear genome dna replicationoccurs once the primer is in placepolymerase switchingsynthesizes lagging strandpol deltasynthesizes leading strandpol epsilonpol alpha and epsilon are replaced by ___ for elongationpol deltaeukaryotic dna complexed with binding proteinschromatin200 base pair nucleosomes wrap around8 histone proteinsInert chromosomal ends that protect intact eukaryotic
chromosomes from improper fusion or degradation
- Long stretches of short repeating sequences preserve the integrity/stability of chromosomestelomerestelomere ___ and a complex of six proteins binds and stabilizes chromosome endst loopstelomere t-loops and a complex of six proteins binds and stabilizes chromosome ends
-- forms the ....shelterin complexR N A serves as template for synthesis of D N A complementtelomeraseTERCtelomerase RNA componentTERTtelomerase reverse transcriptaseis telomerase active in most eukaryotic somatic cellsno___ maintain telomerase activitystem cells and malignant cellsgenetic exchange at equivalent positions along two chromosomes with substantial sequence homologyhomologous recombinationgenetic recombination involves:endonuclease nicking
- strand displacement and pairing with complement
- ligation
- branch migration
- duplex separation— generates characteristicHolliday structureenzymes and proteins involved in homologous recombinationRecA in E.coliRecA in E.coli functionpromotes exchange of reciprocal ssDNA molecules
