D

RecD is lost

RecBC continues to unwind DNA and generate single stranded region

RecBC continues to unwind DNA and generate single stranded region

Integration of lambda into the chromosome of E. coli depends on recognition of the attachment site by a specific integrase protein.

recognition sequence for a specific protein. The protein then initiates the recombination event.

The classic case is the integration of the DNA of bacteriophage lambda (l) into the chromosome of Escherichia coli. Each of these contains a l attachment site (attl). These are designated attBOB' (on the bacterial chromosome) and attPOP' (on the lambda genome). The central core of 15 bases (designated O) of these is identical, but the outermost regions (B and P) differ in size and sequence between host and phage. The core region of both attachment sites is recognized by lambda integrase or Int protein. This makes a staggered double-strand cut in each core sequence. The ends are joined, and the result is that the circle of lambda DNA is inserted into the bacterial chromosome (Fig. 14.08).

attl l attachment site, recognition site on DNA used during integration of lambda DNA into E. coli chromosome integrase Enzyme that inserts a segment of dsDNA into another DNA molecule at a specific recognition sequence. In particular, lambda integrase inserts lambda DNA into the chromosome of E. coli Int protein Same as integrase

FIGURE 14.07 RecA Promotes Strand Invasion

RecA binding stabilizes the unwound single-stranded DNA from Fig. 14.06. The stabilized strand is able to invade the homologous double-stranded DNA forming a triple helix.

recA BINDS SINGLE STRAND

RecA

RecA

Single strand invasion

Homologous DNA

5&W

Homologous DNA

FIGURE 14.07 RecA Promotes Strand Invasion

RecA binding stabilizes the unwound single-stranded DNA from Fig. 14.06. The stabilized strand is able to invade the homologous double-stranded DNA forming a triple helix.

5&W

In fact, the strands are cut and joined one at a time. The first round of cutting and joining gives a Holliday junction and the second round resolves it leading to integration. The arrangement at the crossover is shown in Fig. 14.09. Note that a single crossover event is sufficient for integration of a circular molecule of DNA into another molecule. [Although lambda DNA is linear inside the virus particle, it circularizes upon entering the bacterial cell and before integration (see Ch. 17 for life cycle of lambda).] After integration, lambda DNA is flanked by two hybrid attl sites, attBOP' and attPOB'. Int protein cannot carry out recombination between these hybrid sites and cannot therefore reverse the integration event. Excision of lambda requires Xis protein integration Insertion of a segment of dsDNA into another DNA molecule at a specific recognition sequence lambda attachment site (attl) Recognition site on DNA used during integration of lambda DNA into E. coli chromosome Xis protein Enzyme that reverses DNA integration by removing a segment of dsDNA and resealing the gap leaving behind an intact recognition sequence. Same as excisionase. Not to be confused with Xist RNA involved in X chromosome silencing

FIGURE 14.08 Integration of Lambda DNA—Overview

Bacterial DNA and l phage DNA are aligned at the "O" region of the attachment sites. Int protein induces two double-stranded breaks that are resolved, giving a crossover. Since the two recognition sites are altered in their flanking regions, l cannot be excised by Int alone but needs another protein, known as excisionase or Xis in addition.

Phage

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