9 Геном, плазмиды, вирусы (Лекции), страница 4

DNAunderwinding facilitates strand separation for processes such as transcription or replication. Theplectonemic supercoils in negatively supercoiledDNA in solution are right-handed, and the overallstructure is narrow and extended. An alternativeform called solenoidal supercoiling provides amuch greater degree of compaction, and this formpredominates in the cell.811DNAs that differ only in their linking numberare called topoisomers. The enzymes that underwind and/or relax DNA are called topoisomerases,and they act by catalyzing changes in linking number. There are two classes, type 1 and type 2, whichchange Lk in increments of 1 or 2, respectively.

In abacterial cell, the superhelical density of the DNArepresents a regulated balance between the activities of topoisomerases that increase and decreaselinking number.In the chromatin of eukaryotic cells, the fundamental unit of organization is the nucleosome,which consists of DNA and a protein particle containing eight histones, two copies each of histonesH2A, H2B, H3, and H4. The segment of DNA(about 146 base pairs) wrapped around the proteincore is in the form of a left-handed solenoidal supercoil.

Nucleosomes are organized into 30 nm fibers, and the fibers themselves are extensivelyfolded to provide the 10,000-fold compaction required to fit a typical eukaryotic chromosome intoa cell nucleus. The higher-order folding involvesattachment to a nuclear scaffold that containslarge amounts of histone HI and topoisomerase II.Bacterial chromosomes are also extensively compacted into a structure called a nucleoid, but thechromosome appears to be much more dynamicand irregular in structure than eukaryotic chromatin, reflecting the shorter cell cycle and very activemetabolism of a bacterial cell.812Part IV Information PathwaysFurther ReadingGeneralAlberts, B., Bray, D., Lewis, J., Raff, M., Roberts,K., & Watson, J.D. (1989) Molecular Biology of theCell, 2nd edn, Garland Publishing, Inc., New York.An excellent general reference.Kornberg, A.

& Baker, T.A. (1991) DNA Replication, 2nd edn, W.H. Freeman and Company, NewYork.A good place to start for further information on thestructure and function of DNA.Singer, M. & Berg, P. (1991) Genes and Genomes:A Changing Perspective, University Science Books,Mill Valley, CA.An up-to-date discussion of genes, chromosomestructure, and many other topics.Genes and ChromosomesBlackburn, E.H. (1990) Telomeres: structure andsynthesis. J. Biol. Chem. 265, 5919-5921.Jelinek, W.R. & Schmid, C.W. (1982) Repetitivesequences in eukaryotic DNA and their expression.Annu.

Rev. Biochem. 51, 813-844.Murray, A.W. & Szostak, J.W. (1987) Artificialchromosomes. Sci. Am. 257 (November), 62-68.Novick, R.P. (1980) Plasmids. Sci. Am. 243 (December), 102-127.Sharp, P.A. (1985) On the origin of RNA splicingand introns. Cell 42, 397-400.Ullu, E. & Tschudi, C. (1984) Alu sequences areprocessed 7SL RNA genes. Nature 312, 171-172.Supercoiling and TopoisomerasesBauer, W.R., Crick, F.H.C., & White, J.H. (1980)Supercoiled DNA. Sci. Am. 243 (July), 118-133.Boles, T.C., White, J.H., & Cozzarelli, N.R. (1990)Structure of plectonemically supercoiled DNA.

J.Mol. Biol. 213, 931-951.A study that defines several fundamental featuresof supercoiled DNA.Cozzarelli, N.R., Boles, T.C., & White, J.H. (1990)Primer on the topology and geometry of DNA supercoiling. In DNA Topology and Its Biological Effects (Cozzarelli, N.R. & Wang, J.C., eds), pp. 139184, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY.This provides a more advanced and thorough discussion.Lebowitz, J. (1990) Through the looking glass: thediscovery of supercoiled DNA.

Trends Biochem.Sci. 15, 202-207.A short and interesting historical note.Liu, L.F. (1989) DNA topoisomerase poisons asantitumor drugs. Annu. Rev. Biochem. 58, 351375.A review of eukaryotic topoisomerases and the useof topoisomerase inhibitors in cancer chemotherapy.Wang, J.C. (1985) DNA topoisomerases. Annu.Rev. Biochem. 54, 665-697.Wang, J.C. (1991) DNA topoisomerases: Why somany? J. Biol. Chem. 266, 6659-6662.A good short summary of topoisomerase functions.Chromatin and NucleosomesFilipski, J., Leblanc, J., Youdale, T., Sikorska,M., & Walker, P.R. (1990) Periodicity of DNA folding in higher order chromatin structures. EMBO J.9, 1319-1327.Kornberg, R.D. (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184,868-871.The classic paper that introduced the subunitmodel for chromatin.Richmond, T.J., Finch, J.T., Rushton, B.,Rhodes, D., & Klug, A.

(1984) Structure of thenucleosome core particle at 7A resolution. Nature311, 532-537.van Holde, K.E. (1989) Chromatin, SpringerVerlag, New York.Chapter 23 Genes and Chromosomes813Problems1. How Long Is the Ribonuclease Gene? What isthe minimum number of nucleotide pairs in thegene for pancreatic ribonuclease (124 amino acidslong)? Suggest a reason why the number of nucleotide pairs in the gene might be much larger thanyour answer.2. Packaging ofDNA in a Virus The DNA of bacteriophage T2 has a molecular weight of 120 x 106.The head of the T2 phage is about 210 nm long.Assuming the molecular weight of a nucleotidepair is 650, calculate the length of T2 DNA andcompare it with the length of the T2 head.

Youranswer will show the necessity of very compactpackaging of DNA in viruses (see Fig. 23-1).7. DNA Structure Explain how the underwindingof a B-DNA helix might facilitate or stabilize theformation of Z-DNA.8. Chromatin One of the important early pieces ofevidence that helped define the structure of thenucleosome is illustrated by the agarose gel shownbelow, in which the thick bands represent DNA.

Itwas generated by treating chromatin briefly withan enzyme that degrades DNA, then removing allprotein and subjecting the purified DNA to electrophoresis. Numbers at the side of the gel denote theposition to which a linear DNA of the indicated size(in base pairs) would migrate. What does this geltell you about chromatin structure? Why are theDNA bands thick and spread out rather thansharp?3. The DNA of Phage M13 Bacteriophage M13DNA has the following base composition: A, 23%;T, 36%; G, 21%; C, 20%. What does this informationtell us about the DNA of this phage?4.

Base Composition of cf>X174 DNA Bacteriophage (/>X174 DNA occurs in two forms, singlestranded in the isolated virion and doublestranded during viral replication in the host cell.Would you expect them to have the same base composition? Give your reasons.1000 bp5. Size of Eukaryotic Genes An enzyme present in800 bprat liver has a polypeptide chain of 192 amino acidresidues.

It is coded for by a gene having 1,440 basepairs. Explain the relationship between the number of amino acid residues in this enzyme and thenumber of nucleotide pairs in its gene.6. DNA Supercoiling A covalently closed circularDNA molecule has an Lk of 500 when it is relaxed.Approximately how many base pairs are in thisDNA? How will the linking number be altered (increase, decrease, no change, become undefined) if(a) a protein complex is bound to form a nucleosome, (b) one DNA strand is broken, (c) DNA gyrase is added with ATP, or (d) the double helix isdenatured (base pairs are separated) by heat?200 bpChapter 24 DNA MetabolismTranslesion replication requires DNA polymerase III, as well asthe activities of the UmuC, UmuD, and RecA proteins. The mechanismby which the latter three proteins permit DNA polymerase III to replicate past DNA lesions is not understood.

DNA polymerase II may alsoplay a role in error-prone DNA repair. This polymerase is induced aspart of the SOS response and, unlike DNA polymerase III, is capable oflimited replication past lesions such as AP sites. This enzyme has someof the same subunits as DNA polymerase III.The RecA protein merits some additional discussion because it hasseveral distinct functions (besides mutagenesis) in the bacterial cell.RecA protein is involved in recombination and in the regulation of theSOS response, and in these cases its molecular function is well characterized. The regulation of the SOS response is described in Chapter 27.We now turn to a discussion of genetic recombination.DNA RecombinationThe rearrangement of genetic information in and among DNA molecules encompasses a variety of processes that are collectively placedunder the heading of genetic recombination.

An understanding of howDNA rearrangements occur is finding practical application as scientists explore new methods for altering the genomes of a variety of organisms (Chapter 28).Genetic recombination events fall into at least three generalclasses. Homologous genetic recombination involves genetic exchanges between any two DNA molecules (or segments of the samemolecule) that share an extended region with homologous sequences.The actual sequence of bases in the DNA is irrelevant as long as thesequences in the two DNAs are similar. Site-specific recombinationdiffers in that these exchanges occur only at a defined DNA sequence.DNA transposition is distinct in that it usually involves a short segment of DNA with the remarkable capacity to move from one locationin a chromosome to another.