MCB 502A-2014. Lecture #7Chromosome replication: dnaand pol mutants— Characterization of the dna minus mutants— Isolation of a polA mutant— Isolation and properties of DNA pol II and pol III— The “fork and knife” idea— DNA ligase— The Okazaki experiment— Semidiscontinuous DNA replication (uracil incorporation)?— RNA as a primer for DNA synthesis— PrimaseEnrichment for dna-minus mutants-3— Several important dna mutants wereisolated by this robust protocol.— Later, a brute force automated screenanalyzed 1.4 x 106 mutagenized E. colicolonies to find 2,266 temperature-sensitive mutants, 110 of them defectivein DNA synthesis, but not in proteinsynthesis.— All the various dna-minus E. colimutants, isolated with this enrichmentand the automated screen, fell into threemajor categories, depending on the modeof DNA synthesis inhibition at 42°C.— There were three general tests appliedto characterize DNA synthesis andstability in these mutants.Enrichment for dna-minus mutants-4— Test #1 measured cumulativeincorporation of label over the period ofseveral generations after switch to the non-permissive temperature (28°—>42°C).— The wild type control predictablyaccelerates DNA-label accumulation uponthe temperature shift-up.— The three categories of mutants showeddistinct patterns in this test.— Perhaps the least interesting category, the"inhibited" mutants, never stopped DNAsynthesis, but continued it at a much reducedrate compared with the WT.— The second category immediately stoppedDNA synthesis: the DNA-label accumulationin them plateaued right away at 42°C.TCA-precipitable 3H-dT (DNA)Time (hours)0 1 2WTImmediate-stopDelayed-stop28°–>42°Cinhibited— The third category also stoppedDNA synthesis completely, but onlyafter a delay during which there was asignificant increase in DNA-label.Enrichment for dna-minus mutants-5— Interpretation of the three categories interms of their action at replication forks wasstraightforward:— The "inhibited" mutants identifiedauxiliary replication functions that werehelpful, but not critical, for replication forkprogress.— The immediate stop mutants identifiedfunctions responsible for replication forkprogress. They include mutants in thefollowing loci: dnaB, dnaE, dnaF, dnaG,dnaL, dnaN, dnaQ and dnaX.— The number of genes involved with theprogress of replication forks indicated thecomplex structure of the bacterial replisome.TCA-precipitable 3H-dT (DNA)Time (hours)0 1 2WTImmediate-stopDelayed-stop28°–>42°Cinhibited— The delayed-stop mutants identified functions responsible for replicationinitiation (starting new replication forks). They include mutants in the followingloci: dnaA and dnaC. In contrast to the replisome, initiation seems simple…Enrichment for dna-minus mutants-6— Test #2 monitored changes in the rate ofDNA synthesis after the switch to the non-permissive temperature within onegeneration. The readout for the DNAsynthesis rate was accumulation of DNA-label over the period of one minute.— The inhibited mutants behavedpredictably by showing several fold-reduced, yet constant rate.— The delayed-stop category showedgradually decreasing rate of DNA synthesisupon shift to 42°C over the period of onegeneration before coming to a completestop later on. Their initial assignment as the"initiation defect" was thus confirmed.TCA-precipitable 3H-dT per 1'Time (minutes)0 10 20 30WTImmediate-stopDelayed-stop28°–>42°CinhibiteddnaL— The immediate-stop category, with the exception of dnaL, showed a dramaticfall in the rate of DNA synthesis within a few minutes of shifting to 42°C, also inline with their original assignment as the "replisome defect".Enrichment for dna-minus mutants-7— There was a possibility that the lack ofDNA-label accumulation in some mutantswas due to increased DNA degradation,rather than a defect in DNA synthesis.— This was tested by labeling cells duringgrowth at 28°C, but then removing thelabel just before the shift to 42°C, to revealthe stability of the chromosomal DNA.— The DNA-label was stable in both WTcells and delayed-stop dna mutants.— It was indeed slightly unstable in mostimmediate-stop dna mutants, but notenough to account for the shut-down inaccumulation of DNA-label.TCA-precipitable 3H-dTTime (minutes)0 10 20 30WTImmediate-stopDelayed-stop28°–>42°C, remove the labeldnaL— A single exception, the dnaL mutant, showed a significant rate of DNAdegradation, suggesting instability of DNA synthesized at the non-permissivetemperature.Enrichment for dna-minus mutants-8— Since the dnaL mutant did showsome label accumulation at the non-permissive temperature in the test #2,the experiment with DNA-label stabilitywas repeated with DNA synthesis phasealso at the non-permissive temperature.— It was found that the nascent DNAsynthesized at 42°C was dramaticallyunstable in the dnaL mutant, with itsrate of degradation matching the rate ofsynthesis.— Thus, DnaL was definitely notworking at the replication fork butsomehow was acting to stabilize thenascent DNA.TCA-precipitable 3H-dTTime (minutes)0 10 20 30WTremove the labeldnaLAll at42°C0— Later the DnaL function was shown tocatalyze maturation of the newly-synthesized DNA, rather than its synthesis.Cloning of genes identified by conditional mutations— Identification of the dna conditionalmutants permitted cloning of theresponsible genes by complementationof their lethal phenotype, whichfacilitated biochemical characterizationof the corresponding proteins.— For cloning, one makes a randomlibrary from genomic DNA of wild typecells (randomly-broken chromosomalDNA pieces, linked with an appropriatecloning vector) and transforms adna(Ts) mutant with this library, platingcells at the non-permissive temperatureof 42°C.— Under these conditions, only clonescarrying plasmids with the gene for thefunctional enzyme (= "complementedclones" ) can survive.Concept-in-the-box: Conditionally-lethal mutant offersselection to identify the responsible gene bycomplementation. Indeed, since the inactivated function isessential, the cells can grow and divide only when thefunction is restored (by complementation with the wild typeallele of the gene on a plasmid). This approach will not workonly when the original mutation is a dominant null (themutant enzyme will poison the wild type one), which is rare.Chromosomal DNAReplicationorigin +
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