MCB 502A - 2014. Lecture #4Genomes vs Chromosomes.DNA degradation.— Differential DNA strand labeling as a detection approach. SCE.— The picture of the replicating chromosome (Cairns)Chromosomal organization of genomic DNA— Genome functions— The chromosomal organization of the genome— The differences between prokaryotic and eukaryotic chromosomesDNA degradation— The basic configurations of the natural DNA molecules— Gel electrophoresis— Southern blotting (detection of DNA degradation in vivo)— TCA precipitation— Major nucleases of E. coliDifferential DNA strand labelingas a detection approach—What is wrong with this scheme?The bivalent was replicated conservatively!Sister-chromatidexchange (SCE)-1— Besides confirming the semiconservativemode of DNA replication, Taylor’s resultsilluminated a new phenomenon.— Occasionally, after the 2nd replicationround, some radioactivity would be found inthe otherwise non-radioactive chromosome.— Remarkably, in all these cases, theotherwise radioactive chromosome of thebivalent would be always non-radioactive inthe corresponding segment!— This observation suggested a reciprocalswapping of chromosomal arms,subsequently called “sister-chromatidexchange” (SCE).SCESister-chromatid exchange (SCE)-2— Due to the low density of radioactive labeling, theoriginal observation of Taylor was not highlyconvincing.— However, sister-chromatid exchange in cells ofhigher eukaryotes (higher plants, mammals) waseventually fully confirmed by chemical methods ofchromosomal staining, in particular by the methodemploying bromodeoxyuridine (BrdU), an analog ofthymidine.— Cells are grown in the presence of BrdU for twogenerations, the second anaphase is blocked, thechromosomes are spread and then stained with BrdU-specific dyes.— This staining allows good distinction between thechromatid in the bivalent in which only one DNAstrand is BrdU-labeled and its sister chromatid inwhich both DNA strands are BrdU-labeled.Sister-chromatid exchange (SCE)-3— Sister-chromatid exchanges areseen as reciprocal changes in thelabeling pattern between the twosister chromosomes, still attached toeach other at the centromere.— We now know that sister-chromatid exchanges indicateinstances of recombinationalrepair of double-strand breaks(more on it later).— Multiple exchanges in certainmutants produce the pattern called“harlequin chromosomes”,characteristic of defects in the DNAmetabolism, associated with somecancer-predisposition syndromes inhumans.Sister-chromatid exchange (SCE)-4Normal cells Bloom'ssyndrome cellsNormal cells treatedwith a DNA-damaging agentMMShttp://www.pnas.org/content/98/15/8196/F1.expansion.htmlSister-chromatid exchange (SCE)-5— Can one apply this spectaculardifferential labeling to detect sister-chromatid exchange in prokaryotes?— Not directly, because 1) prokaryoticchromosomes are small and cannot beseen in detail under the light microscope;2) unlike eukaryotic sister chromatids atthe metaphase of mitosis, prokaryoticsister chromatids are never condensedinto connected compact bodies at anypart of the cell cycle.— Therefore, direct visualization is notavailable in prokaryotes.— However, detection of the DNAstrand exchange by differential strandlabeling is possible in bacteria, ifcombined with other methods.Sister-chromatid exchange (SCE)-6— For example, Walter Steiner and PeterKuempel suspected a site-specificrecombination-catalyzed strand exchange in aparticular region of the E. coli chromosome.Frank Stahl Walter SteinerSister-chromatidexchange (SCE)-6— To detect this exchange, they grewcells in a heavy medium, supplementedwith 15N and 13C, and then switchedthem to a light, 14N 12C-containingmedium for one generation.— DNA duplexes became half-light,half-heavy.— The researchers sheared the DNA toa relatively low MW, separated the twostrands of the hybrid duplexes inalkaline (DNA denaturing) densitygradient and observed two peaks,corresponding to the heavy and the lightDNA strands.xxdifhttp://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.00651.x/full+Sister-chromatidexchange (SCE)-7— They collected fractions covering these peaks, spottedthem on a nylon membrane and hybridized with a probecentered on the small DNA region (dif), in which theysuspected a frequent site-specific strand exchange.— Such an exchange would generate strands, in which heavyDNA is joined with light DNA at the point of exchange.— The Dot-hybridization with a region-specific probe indeedrevealed three peaks instead of two!— Now there was an additional peak of intermediate density,exactly what was expected if there were a strand exchange inthis sequence.— As a negative control, the dot-hybridization with probesaway from the region revealed only two peaks, the heavy oneand the light one.http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.00651.x/fullSister-chromatid exchange (SCE)-8— These are powerfulexperimental approaches todetect strand exchange, whichexploit the fact that DNAreplicates semiconservatively.The picture of the replicatingchromosome (Cairns)-1— You have probably noticed, that the demonstration of semiconservativereplication did not resolve the issue of DNA duplex rotation during replication, —it just confirmed that bacterial cells are capable of unwinding over 450,000 turnsin 45 minutes (10,000 revolutions per minute!).— It seemed impossible that a single replication point could rotate with such aspeed (~10x the rotation speed of a propeller of a cruising turboprop engine).— Therefore, it was proposed that the actualnumber of replication points is significant, so thisrotation is spread among many points goingconcurrently.— For example, if there were 100 replication pointsin the E. coli chromosome, the speed of DNArotation during replication at any single point wouldbe only 100 revolutions per minute — quite adifferent story.The picture of the replicatingchromosome (Cairns)-2— At that time nothing was known not only about how the E. colichromosome replicates, but also about how the genomic DNA ofE. coli is organized into its chromosome.— Genetic data on marker transfer suggested a single circularchromosome of an enormous size, whereas physicalmeasurements of the DNA pieces extracted from cells of anyorganism showed that DNA is of the uniform size of about 5-10kbp.— (Only later it was found that this uniform size is produced byshearing in syringes, used to distribute and
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