Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 77
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The centromeric DNA is containedin a structure (the centromeric core particle) that contains more DNA than a typical yeast nucleosome core particle(which contains 160 base pairs) and is larger. This structure is responsible for the resistance of centromeric DNA toDNase. The spindle fiber is believed to be attached directly to this particle (Figure 6.25B).The base-sequence arrangement of the yeast centromeres is not typical of other eukaryotic centromeres. In highereukaryotes, the chromosomes are about 100 times as large as yeast chromosomes, and several spindle fibers areusually attached to each region of the centromere.
Furthermore, the centromeric regions of the chromosomes ofmany higher eukaryotes contain large amounts of heterochromatin, consisting of many higher eukaryotes containlarge amounts of heterochromatin, consisting of repetitive satellite DNA, as described in Section 6.7.
For example,the centromeric regions of human chromosomes contain a tandemly repeated DNA sequence of about 170 basepairs called the alpha satellite (Figure 6.26). The number of alpha-satellite copies in the centromeric region rangesfrom 5000 to 15,000, depending on the chromosome. The DNA sequences needed for spindle fiber attachment maybe interspersed among the alpha-satellite sequences, but whether the alpha-satellite sequences themselvescontribute to centromere activity is unknown.Molecular Structure of the TelomereEach end of a linear chromosome is composed of a special DNA-protein structure called a telomere that isessential for chromosome stability.
Genetic and microscopicFigure 6.25A yeast centromere. (A) Diagram of centromeric DNA showing the majorregions (CDE1 through CDE4) common to all yeast centromeres. Theletter R stands for any purine (A or G), and the letter N indicates anynucleotide. Inverted-repeat segments in region 3 are indicated by arrows.The sequence of region CDE4 varies from one centromere to the next.(B) Positions of the centromere core and the nucleosomes on the DNA.The DNA is wrapped around histones in the nucleosomes, but thedetailed organization and composition of the centromere core areunknown.[After K. S. Bloom, M.
Fitzgerald-Hayes, and J. Carbon. 1982.Cold Spring Harbor Symp. Quant. Biol., 47: 1175.]Page 248Figure 6.26Hybridization of human metaphase chromosomes (red) with alpha-satellite DNA.The yellow areas result from hybridization with the labeled DNA. The sites ofhybridization of the alpha satellite coincide with the centromeric regions of all 46chromosomes.[Courtesy of Paula Coelho and Claudio E. Sunkel.]observations first indicated that telomeres are special structures. In Drosophila, Hermann J. Muller found thatchromosomes without ends could not be recovered after chromosomes were broken by treatment with x rays.
Inmaize, Barbara McClintock observed that broken chromosomes frequently fuse with one another and form newchromosomes with abnormal structures (often having two centromeres). As we saw in Chapter 5, DNApolymerases cannot initiate DNA synthesis but instead require an RNA primase, so at least one end of eachchromosome must have a short (8 to 12 nucleotides) stretch of single-stranded DNA that remains after the RNAprimer at the tip has been removed.
It turns out that both ends of linear chromosomes terminate in a stretch ofsingle-stranded DNA.Telomeric DNA in most organisms consists of tandem repeats of simple sequences such as 5'-TTAGGG-3'. Thesespecial telomere DNA sequences are added to the ends of eukaryotic chromosomes by an enzyme calledtelomerase. The substrate for the telomerase is a telomere addition sequence consisting of a short repetitivesequence. In mammals, the telomere addition sequence consists of many tandem repeats of 5'-TTAGGG-3'. In theciliate protozoan Tetrahymena, it consists of repeats of the similar sequence 5'-TTGGGG-3'.
Relatively few copiesof the repeat are necessary to prime the telomerase to add additional copies and form a telomere. Remarkably, thetelomerase enzyme incorporates an essential RNA molecule, called a guide RNA, that contains sequencescomplementary to the telomere repeat and that serves as a template for telomere synthesis and elongation. Forexample, the Tetrahymena guide RNA contains the sequence 3'-AACCCCAAC-5'. The guide RNA undergoes basepairing with the telomere repeat and serves as a template for telomere elongation by the addition of more repeatingunits (Figure 6.27).
The complementary DNA strand of the telomere is analogous to the lagging strand at areplication fork and is synthesized by DNA polymerase in the usual manner with thePage 249Connection Telomeres: The Beginning of the EndCarol W.
Greider and Elizabeth H.Blackburn 1987University of California,Berkeley, CaliforniaThe Telomere Terminal Transferase ofTetrahymena Is a Ribonucleoproteinenzyme with Two Kinds of Primer SpecificityWhat a wonderful surprise that an RNA is a key ingredient in the formation of telomeres! Two limitations of DNApolymerase are that it requires a primer oligonucleotide and that it can elongate a DNA strand only at the 3' end.The limitations imply that the 3' end of a DNA strand should become progressively shorter with each round ofreplication, owing to the need for a primer at that extremity.
(Chromosomes without proper telomeres do, in fact,become progressively shorter.) The organism used in this study, Tetrahymena, is a ciliated protozoan. Each cellhas a specialized type of nucleus called a macronucleus that contains many hundreds of small chromosomes. Thelevel of telomerase activity is high because each of these tiny chromosomes needs a pair of telomeres. Theconvenience of using Tetrahymena for the study of telomere function illustrates a principle that runs through thehistory of genetics: Breakthroughs often come from choosing just the right organism to study.
The authorsspeculate about the possible presence of a "guide" RNA in the telomerase. They were exactly right.Precise recognition of nucleic acids is often carried out by enzymes that contain both RNA and proteincomponents. For some of these ribonucleoproteins (RNPs), the RNA components provide specificity to the reactionby base pairing with the substrate.
The recognition that RNA can act catalytically has led to the increase in thenumber of known RNP-catalyzed reactions. We report here that an RNP is involved in synthesizing the telomericsequences. . . it is tempting to speculate that the RNA component of the telomerase might be involved indetermining the sequence of the telomeric repeats that are synthesized . . .found at the ends of Tetrahymena macronuclear chromosomes. . . . We have previously reported the identificationof an activity in Tetrahymena cell extracts that adds telomeric repeats onto appropriate telomeric sequence primersin a nontemplated manner. .
. . Repeats of the Tetrahymena telomeric sequence TTGGGG are added, 1 nucleotideat a time, onto the 3' end of the input primer. . . . We have begun characterizing and purifying the telomeraseenzyme in order to investigate the mechanisms controlling the specificity of the reaction. . .
. We propose that theRNA component(s) of telomerase may play a role in specifying the sequence of the added telomeric TTGGGGrepeats, in recognizing the structure of the G-rich telomeric sequence primers, or both. . . . In the course ofpurifying the telomerase from crude extracts, we noted a marked sensitivity to salts [in which high] concentrationsinactivated the telomeric elongation activity. The salt sensitivity and the large size of the enzyme suggested that thetelomerase may be a complex containing a nucleic acid component. To test whether the telomerase contained anessential nucleic acid, we treated active fractions with either micrococcal nuclease or RNAase A.
The nucleaseactivity of each of these enzymes abolished the telomeric elongation activity. . . . These experiments suggest thatthe telomerase contains an essential RNA component. . . . The RNA of telomerase may simply provide a scaffoldfor the assembly of proteins in the active enzyme complex; however, . . . it is tempting to speculate that the RNAcomponent of the telomerase might be involved in determining the sequence of the telomeric repeats that aresynthesized and/or the specific primer recognition. If the RNA of telomerase contains the sequence CCCCAA, thissequence could act as an internal guide sequence.Source: Cell 51:887–898help of an RNA primer. In the telomeric regions in most eukaryotes, there are also longer, moderately repetitive DNAsequences just preceding the terminal repeats.
These sequences differ among organisms and even among differentchromosomes in the same organism.What limits the length of a telomere? In most organisms the answer is unknown. In yeast, however, a protein calledRaplp has been identified that appears to be important in regulating telomere length.
The Raplp protein binds to theyeast telomere sequence. Molecules of Raplp bind to the telomere sequence as it is being elongated until about 17Raplp molecules have been bound. At this point, telomere elongation stops, probably because the accumulation ofRaplp inhibits telomerase activity. Because each Raplp molecule binds to approximately 18 base pairs of thetelomere, the predicted length of a yeast telomere is 17 × 18 = 306 base pairs, which is very close to the valueobserved.
Additional evidence for the role of Raplp comes from mutations in the RAP l gene producing a protein thacannot bind to telomere sequences; in these mutantPage 250Figure 6.27Telomere formation in Tetrahymena. The telomerase enzyme contains an internalRNA with a sequence complementary to the telomere repeat. The RNA undergoes basepairing with the telomere repeat and serves as a template for telomere elongation.The newly forming DNA strand is produced by DNA polymerase.strains, massive telomere elongation is observed.Telomeric sequences found in a variety of organisms are shown in Table 6.2.
The table does not includeDrosophila because its telomeres, rather than consisting of simple repeats as in most other organisms, arecomposed of specialized transposable elements. Each telomere sequence in Table 6.2 in written as if it were at theleft-hand end of a chromosome. Both strands are written in the 5'-to-3' direction. The top strand is the 5' end of thetelomere, and the bottom strand is the 3' end of the telomere. It is the 3' end of the bottom strand (in each entry, the3' end is at the far right) that is elongated by the telomerase and that is single-stranded.
Runs of nucleotides areindicated by subscripts; for example, the notation C3TA2/T2AG3 for vertebrate telomeres means the sequenceIn many cases, there is variation in the length of a run; for example, C1–8 means variation ranging from as few asone C to as many as eight C's from one repeat to the next. There is also occasional variation in which nucleotideoccupies a given position, which is indicated by small stacked letters.There is a strong tendency for the elongated strand of the telomere repeats in Table 6.2 to be rich in guaninenucleotides. The guanine bases are special in that they have the capacity to hydrogen-bond to one another in avariety of ways. When the elongated 3' strand of the telomere folds back upon itself, the guanine bases can evenpair to give the G-quartet structure illustrated in Figure 6.28.
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