H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 44
Текст из файла (страница 44)
As a consequence, the atoms on the edgesof each base within these grooves are accessible from outside the helix, forming two types of binding surfaces. DNAbinding proteins can “read” the sequence of bases in duplexDNA by contacting atoms in either the major or the minorgrooves.In addition to the major B form, three additional DNAstructures have been described. Two of these are comparedto B DNA in Figure 4-4.
In very low humidity, the crystallographic structure of B DNA changes to the A form; RNADNA and RNA-RNA helices exist in this form in cells and invitro. Short DNA molecules composed of alternating purinepyrimidine nucleotides (especially Gs and Cs) adopt an alternative left-handed configuration instead of the normalright-handed helix. This structure is called Z DNA because4.1 • Structure of Nucleic Acidsthe bases seem to zigzag when viewed from the side. Someevidence suggests that Z DNA may occur in cells, althoughits function is unknown.
Finally, a triple-stranded DNAstructure is formed when synthetic polymers of poly(A) and(b) A DNA(c) Z DNA3.6 nm(a) B DNA▲ FIGURE 4-4 Models of various known DNA structures.The sugar-phosphate backbones of the two strands, which areon the outside in all structures, are shown in red and blue; thebases (lighter shades) are oriented inward. (a) The B form of DNAhas ≈10.5 base pairs per helical turn. Adjacent stacked base pairsare 0.36 nm apart. (b) The more compact A form of DNA has11 base pairs per turn and exhibits a large tilt of the base pairswith respect to the helix axis.
(c) Z DNA is a left-handed doublehelix.TATA box–binding protein▲ FIGURE 4-5 Bending of DNA resulting from proteinbinding. The conserved C-terminal domain of the TATA box–binding protein (TBP) binds to the minor groove of specific DNAsequences rich in A and T, untwisting and sharply bending thedouble helix. Transcription of most eukaryotic genes requiresparticipation of TBP. [Adapted from D.
B. Nikolov and S. K. Burley, 1997,Proc. Nat’l. Acad. Sci. USA 94:15.]105polydeoxy(U) are mixed in the test tube. In addition, homopolymeric stretches of DNA composed of C and Tresidues in one strand and A and G residues in the other canform a triple-stranded structure by binding matching lengthsof synthetic poly(CT).
Such structures probably do notoccur naturally in cells but may prove useful as therapeuticagents.By far the most important modifications in the structureof standard B-form DNA come about as a result of proteinbinding to specific DNA sequences. Although the multitudeof hydrogen and hydrophobic bonds between the bases provide stability to DNA, the double helix is flexible about itslong axis. Unlike the helix in proteins (see Figure 3-3),there are no hydrogen bonds parallel to the axis of the DNAhelix. This property allows DNA to bend when complexedwith a DNA-binding protein (Figure 4-5).
Bending of DNAis critical to the dense packing of DNA in chromatin, theprotein-DNA complex in which nuclear DNA occurs in eukaryotic cells (Chapter 10).DNA Can Undergo ReversibleStrand SeparationDuring replication and transcription of DNA, the strands ofthe double helix must separate to allow the internal edges ofthe bases to pair with the bases of the nucleotides to be polymerized into new polynucleotide chains. In later sections, wedescribe the cellular mechanisms that separate and subsequently reassociate DNA strands during replication andtranscription. Here we discuss factors influencing the in vitroseparation and reassociation of DNA strands.The unwinding and separation of DNA strands, referredto as denaturation, or “melting,” can be induced experimentally by increasing the temperature of a solution of DNA.
Asthe thermal energy increases, the resulting increase in molecular motion eventually breaks the hydrogen bonds andother forces that stabilize the double helix; the strands thenseparate, driven apart by the electrostatic repulsion of thenegatively charged deoxyribose-phosphate backbone of eachstrand. Near the denaturation temperature, a small increasein temperature causes a rapid, near simultaneous loss of themultiple weak interactions holding the strands togetheralong the entire length of the DNA molecules, leading to anabrupt change in the absorption of ultraviolet (UV) light(Figure 4-6a).The melting temperature Tm at which DNA strands willseparate depends on several factors.
Molecules that containa greater proportion of G·C pairs require higher temperatures to denature because the three hydrogen bonds in G·Cpairs make these base pairs more stable than A·T pairs,which have only two hydrogen bonds. Indeed, the percentageof G·C base pairs in a DNA sample can be estimated from itsTm (Figure 4-6b).
The ion concentration also influences theTm because the negatively charged phosphate groups in the106CHAPTER 4 • Basic Molecular Genetic Mechanisms(a)(b)100Single-strandedDNAPercentage of G•C pairsAbsorption of 260-nm light1.00.75Double-strandedDNA80604020Tm0.575808590Temperature (°C)0708090100110Tm (°C)▲ EXPERIMENTAL FIGURE 4-6 The temperature at whichDNA denatures increases with the proportion of GC pairs.(a) Melting of doubled-stranded DNA can be monitored by theabsorption of ultraviolet light at 260 nm.
As regions of doublestranded DNA unpair, the absorption of light by those regionsincreases almost twofold. The temperature at which half thebases in a double-stranded DNA sample have denatured isdenoted Tm (for temperature of melting). Light absorption bysingle-stranded DNA changes much less as the temperature isincreased. (b) The Tm is a function of the GC content of theDNA; the higher the G+C percentage, the greater the Tm.two strands are shielded by positively charged ions.
Whenthe ion concentration is low, this shielding is decreased, thusincreasing the repulsive forces between the strands and reducing the Tm. Agents that destabilize hydrogen bonds, suchas formamide or urea, also lower the Tm. Finally, extremes ofpH denature DNA at low temperature. At low (acid) pH, thebases become protonated and thus positively charged, repelling each other. At high (alkaline) pH, the bases lose protons and become negatively charged, again repelling eachother because of the similar charge.The single-stranded DNA molecules that result from denaturation form random coils without an organized structure.
Lowering the temperature, increasing the ionconcentration, or neutralizing the pH causes the two complementary strands to reassociate into a perfect double helix.The extent of such renaturation is dependent on time, theDNA concentration, and the ionic concentration. Two DNAstrands not related in sequence will remain as random coilsand will not renature; most importantly, they will not inhibitcomplementary DNA partner strands from finding eachother and renaturing. Denaturation and renaturation ofDNA are the basis of nucleic acid hybridization, a powerfultechnique used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in amixture containing numerous different DNA sequences (seeFigure 9-16).cells, and in chloroplasts, which are present in plants andsome unicellular eukaryotes.Each of the two strands in a circular DNA moleculeforms a closed structure without free ends.
Localized unwinding of a circular DNA molecule, which occurs duringDNA replication, induces torsional stress into the remaining portion of the molecule because the ends of the strandsare not free to rotate. As a result, the DNA molecule twistsback on itself, like a twisted rubber band, forming supercoils (Figure 4-7b). In other words, when part of the DNAhelix is underwound, the remainder of the molecule becomes overwound.
Bacterial and eukaryotic cells, however,contain topoisomerase I, which can relieve any torsionalstress that develops in cellular DNA molecules during replication or other processes. This enzyme binds to DNA atrandom sites and breaks a phosphodiester bond in onestrand. Such a one-strand break in DNA is called a nick.The broken end then winds around the uncut strand, leading to loss of supercoils (Figure 4-7a). Finally, the same enzyme joins (ligates) the two ends of the broken strand.Another type of enzyme, topoisomerase II, makes breaks inboth strands of a double-stranded DNA and then religatesthem.
As a result, topoisomerase II can both relieve torsional stress and link together two circular DNA moleculesas in the links of a chain.Although eukaryotic nuclear DNA is linear, long loops ofDNA are fixed in place within chromosomes (Chapter 10).Thus torsional stress and the consequent formation of supercoils also could occur during replication of nuclear DNA.As in bacterial cells, abundant topoisomerase I in eukaryoticnuclei relieves any torsional stress in nuclear DNA thatwould develop in the absence of this enzyme.Many DNA Molecules Are CircularMany prokaryotic genomic DNAs and many viral DNAs arecircular molecules. Circular DNA molecules also occur inmitochondria, which are present in almost all eukaryotic4.1 • Structure of Nucleic Acids(a) Supercoiled(b) Relaxed circle107 EXPERIMENTAL FIGURE 4-7 DNAsupercoils can be removed by cleavageof one strand.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
