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Terminal inverted repeat Flanking direct repeat

Terminal inverted repeat Flanking direct repeat

11.17 Many transposable elements have common characteristics. (a) Most transposable elements generate flanking direct repeats on each side of the point of insertion into target DNA. Many transposable elements also possess terminal inverted repeats. (b) These representations of direct and indirect repeats are used in illustrations throughout this chapter.

Transposable element

Terminal inverted repeat Flanking direct repeat transposable element is joined to single-stranded ends of the target DNA; and (3) DNA is replicated at the singlestrand gaps.

Mechanisms of Transposition

Some transposable elements transpose through DNA intermediates, whereas others use RNA intermediates. Among those that transpose through DNA, transposition may be replicative or nonreplicative. In replicative transposition, a new copy of the transposable element is introduced at a new site while the old copy remains behind at the original site; the number of copies of the transposable element increases. In nonreplicative transposition, the transposable element excises from the old site and inserts at a new site without any increase in the number of its copies. Nonreplicative transposition requires replication of only the few nucleotides that constitute the direct repeats.

Replicative transposition Replicative transposition, sometimes called copy-and-paste transposition, can be either between two different DNA molecules or between two parts of the same DNA molecule. I FIGURE 11.18 summarizes the steps of transposition between two circular DNA molecules. Before transposition (see Figure 11.18a), the transposable element is on one molecule. In the first step, the two DNA molecules are joined, and the transpos-able element is replicated, producing the cointegrate structure that consists of molecules A + B fused together with two copies of the transposable element (see Figure 11.18b). In a moment, we'll see how the copy is produced, but let's first look at the second step of the replicative transposition process. After the cointegrate has formed, crossing over at regions within the transposable elements produces two molecules, each with a copy of the transposable element

(see Figure 11.18c). This second step is known as resolution of the cointegrate.

How are the steps of replicative transposition (cointe-grate formation and resolution) brought about? Cointegrate formation requires four events. First, a transposase enzyme (often encoded by the transposable element) makes singlestrand breaks at each end of the transposable element and on either side of the target sequence where the element inserts ( FIGURE 11.19 a and b). Second, the free ends of the transposable element attach to the free ends of the target sequence ( FIGURE 11.19c). Third, replication takes place on the single-stranded templates, beginning at the 3' ends of the single strands and proceeding through the transposable element ( FIGURE 11.19d and e). This replication creates the cointegrate, with its two copies of both the transposable element and the sequence at the target site, which is now on one side of each copy ( FIGURE 11.19f). The enzymes that perform the replication and ligation functions are cellular enzymes that function in replication and DNA repair. Fourth, after the cointegrate has formed, it undergoes resolution, which requires crossing over between sites located within the transposon. Resolution gives rise to two copies of the transposable element ( FIGURE 11.19g). The resolution step is brought about by resolvase enzymes (encoded in some cases by the transposable element and in other cases by a cellular gene) that function in homologous recombination.

Nonreplicative transposition In nonreplicative transposition, the transposable element moves from one site to another without replication of the entire transposable element, although short sequences in the target DNA are replicated, generating flanking direct repeats. Sometimes referred to as cut-and-paste transposition, nonreplicative transposition requires only that the transposable element

(c) Resolution

(c) Resolution

l"| Before transposition takes place, a single copy of the transposable element is found on only one molecule

4 .results in two separate molecules, each with a copy of the transposable element.

l"| Before transposition takes place, a single copy of the transposable element is found on only one molecule

4 11.18 Replicative transposition increases the number of copies of the transposable element.

4 .results in two separate molecules, each with a copy of the transposable element.

l'| One copy of the transposable element is present.

^ A transposase enzyme makes singlestrand breaks at each end of the transposable element...

l'| One copy of the transposable element is present.

Target sequence

. and at the ends of the target sequence.

. and at the ends of the target sequence.

4l The free ends of the transposable element attach to the free ends of the target sequence.

5j Replication takes place on the single-stranded templates, beginning at the free ends of the single strands.

4l The free ends of the transposable element attach to the free ends of the target sequence.

5j Replication takes place on the single-stranded templates, beginning at the free ends of the single strands.

11.19 Replicative transposition requires single-strand breaks, replication, and resolution.

and the target DNA be cleaved and joined together. Cleavage requires a transposase enzyme produced by the transposable element. The joining of the transposable element and target DNA is probably carried out by normal replication and repair enzymes. If a transposable element moves by nonreplicative transposition, how does it increase in copy number in the genome? The answer comes from examining the fate of the original site of the element. After excision, a break will be left at the original insertion site. Such breaks are harmful to the cell, and so they are repaired efficiently (see Chapter 17). One common method of repair is to copy sequence information from a homologous template; the sister chromatid is the preferred template for this type of repair. Before transposition, both sisters will have a copy of the transposable element. After excision from one chro-matid, repair of the break can result in copying the transpos-able element sequence off the sister chromatid. Thus, the transposable element is moved from the original site to a new site, but a copy is restored to the original site by DNA repair mechanisms.

Transposition through an RNA intermediate Eukaryotic transposable elements that transpose through RNA intermediates are called retrotransposons. A retrotransposon in DNA (FIGURE 11.20a) is first transcribed into an RNA sequence (FIGURE 11.20b), which may be processed. The processed RNA undergoes reverse transcription by a reverse tran-scriptase enzyme to produce a double-stranded DNA copy of the RNA (IFigure 11.20c). Staggered cuts are made in the target DNA (< FIGURE 11.20d), and the DNA copy of the retrotransposon inserts into the genome ( FIGURE 11.20e). Replication fills in the short gaps produced by the staggered cuts, generating flanking direct repeats on both sides of the retrotransposon.

Concepts]"

Transposition may be through either a DNA or an RNA intermediate. In replicative transposition, a new copy of the transposable element inserts in a new location and the old copy stays behind; in nonreplicative transposition, the old copy excises from the old site and moves to a new site. Transposition through an RNA intermediate requires reverse transcription, in which a retrotransposon is transcribed into RNA, the RNA is copied into DNA, and the new DNA copy is integrated into the target site.

The Mutagenic Effects of Transposition

Because transposable elements may insert into other genes and disrupt their function, transposition is generally muta-genic. In fact, more than half of all spontaneously occurring mutations in Drosophila result from the insertion of a trans-posable element in or near a functional gene. Although most of these mutations are detrimental, transposition may occasionally activate a gene or change the phenotype of the cell in a beneficial way. Additionally, a transposable element may carry information that benefits the cell, such as antibiotic resistance conferred by genes carried on bacterial trans-posable elements.

In 1991, Francis Collins and his colleagues discovered a 31-year-old man with neurofibromatosis caused by a transposition of the Alu sequence. Neurofibromatosis is a disease

111.20 (opposite page) Retrotransposons transpose through RNA intermediates.

| .and proceeding through the transposable element and the target sequences.

l\ .to produce the cointegrate, with two copies of the transposable element and two copies of the target sequence.

^ Crossing over between sites within the transposable element.

|9| .gives rise to two separate copies of the transposable element.

| .and proceeding through the transposable element and the target sequences.

l\ .to produce the cointegrate, with two copies of the transposable element and two copies of the target sequence.

^ Crossing over between sites within the transposable element.

Fo| The new copy is flanked by direct repeats of the target sequence.

(a) Flanking Retrotransposon Flanking direct direct repeat repeat

(a) Flanking Retrotransposon Flanking direct direct repeat repeat

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