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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).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 <f)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.5. Size ofEukaryotic Genes An enzyme present inrat 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?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?1000 bp—800 bp—600 bp—400 bp—200 bp—C H A P T E RDNA MetabolismAs the repository of genetic information, DNA occupies a unique andcentral place among biological macromolecules.
The nucleotide sequences of DNA ultimately describe the primary structures of all cellular RNAs and proteins, and through enzymes can indirectly affect thesynthesis of all other cellular constituents, determining the size,shape, and function of every living thing.The structure of DNA is a marvelous device for the stable storageof genetic information. The phrase "stable storage," however, conveys astatic and incomplete picture of the biochemical role of DNA in the cell.A proper description of DNA function must also explain how that information is transmitted from one generation of cells to the next. Theterm "DNA metabolism" can be used to describe the process by whichfaithful copies of DNA molecules are made (replication), along with theprocesses that affect the structure of the information within (repairand recombination). Together they are the focus of this chapter.Perhaps more than any other factor, it is the requirement for anexquisite degree of accuracy that shapes these processes.
At the level ofjoining one nucleotide to the next, the chemistry of DNA replication issimple and elegant, almost deceptively so. But as we will see, the synthesis of all macromolecules that contain information involves complexdevices to ensure that the information is transmitted intact. If leftuncorrected, errors in DNA synthesis can have dire consequences because they are essentially permanent. The enzymes that synthesizeDNA must copy DNA molecules that often contain millions of bases,and they do so with great fidelity and speed. They must also act on aDNA substrate that is highly compacted and bound with other proteins. The enzymes that catalyze the formation of phosphodiesterbonds are therefore only part of an elaborate system involving myriadproteins and enzymes.The importance of maintaining the integrity of the informationstored in DNA is underscored when the discussion turns to repair.
Asdetailed in Chapter 12, DNA is susceptible to many types of damagingreactions. Though generally slow, they are nevertheless significantbecause of the very low biological tolerance for changes in DNA sequence. DNA is the only macromolecule for which repair systems exist,and their number, diversity, and complexity reflect the wide range ofinsults to which a DNA molecule is subject.The processes by which genetic information is rearranged, collectively called recombination, seem to belie the principles just established. If the integrity of the genetic information is paramount, whyrearrange it? One explanation seems to be the need for maintaining a814Chapter 24 DNA Metabolismlevel of genetic diversity by providing new combinations of alleles, thealternative forms of a single gene. Even without this explanation, however, recombination is not really so renegade a set of processes.
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