L

aire fnt IHF

-ihf

FIGURE 11-9 Recombination sites involved in k integration and excision showing the important sequence elements. C, C, 6, and B* are the care Wet binding sites. The additional protein binding sites aie on atiP and flank the c and C .sites. These re grans are called the "arms" the sequences on the left are called the F arm and those on the nghr are called the P' arm. (Vie small purple boxes labeled Pr, Pi, and P |' are the arm Mnt binding sites. Sites matted H are the IHFbmding sites, and sites marked X are the sites which bind Xis. I- is the site bound by f tsr another architectural protein not discussed further here. The gray regions are the crossover regions. For clarity, Mnt is not shown bound to the core sites. Note that not aB protein binding sites are filled during eitfier integrative or erosive recombination. ftfiei lecombindtion, tlie P arm is pari of atti whereas, the P' arm becomes part of attR.

attR

H"

figure 11-10 Model for IHF bending DNA to bring DNA-binding sites together.

The Mnt and IHF binding sites from the P' arm of ait!3 are shown IhF binding tn the H site bends the DNA to allow one molecule of Mrit to bind both the P,r and C sites. The break in the DMA within the H' site reflects a nick that was present in the ONA used for structural analysis of the 1HF-DNA complex. (Source: From Rice P. et at 1996. Crystal structure of an I Hi-DMA Complex. Cell 87: 13OA Copyright © 1996, with permission from Elsevier.)

figure 11-10 Model for IHF bending DNA to bring DNA-binding sites together.

The Mnt and IHF binding sites from the P' arm of ait!3 are shown IhF binding tn the H site bends the DNA to allow one molecule of Mrit to bind both the P,r and C sites. The break in the DMA within the H' site reflects a nick that was present in the ONA used for structural analysis of the 1HF-DNA complex. (Source: From Rice P. et at 1996. Crystal structure of an I Hi-DMA Complex. Cell 87: 13OA Copyright © 1996, with permission from Elsevier.)

a centra} core segment (approximately 30 hp). These core recombination sites each consist of two Mnt binding sites and a crossover region where strand exchange occurs (as described above). Whereas ottB consists only of this central core region, attP is much longer (240 bp) and carries numerous additional protein binding sites.

Flanking each side of the core region of attP are UNA regions knowrn as the "arms." These arms carry a variety of protein binding sites, including additional sites bound by Xlnt (labeled as Pi, P2, and P\ in Figure 11-9). Mnt is an unusual protein because it has two domains involved in sequence-specific DNA binding: one domain binds to the arm recombinase recognition sites and the other binds to the core recognition sites. In addition, the arms of attP carry sites bound by several architectural proteins. Binding of these proteins governs the directionality and efficiency of recombination.

Integration requires attR, attP, Mnt, and an architectural protein called integration host factor (IHF). IHF is a sequence-dependent DNA-binding protein thai introduces large bends (> 160') in DNA (Figure 11-10), The arms of attP carry throe IHF sites (labeled H,, HZf and H' in Figure 11-9). The function of IHF is to bring together the Xlnt sites on the DNA arms (where Mnt binds strongly) with the sites present at the central core (where it binds only weakly) but where it must bind to catalyze recombination.

When recombination is complete, the circular phage genome is stably inlegrated into the host chromosome. As a resull, two new, hybrid sites are generated at the junctions between the phage and the host DNA. These sites are called cittL (left) and attR (right) (see Figure 11-9). Both of these sites conlain the core region, but the two arm regions are now separated from one another (see the location of the P and P' regions in Figure 11-9). Thus, neither of the two core regions in this new arrangement is competent to assemble an active Mnt recombinase complex via the mechanism that was used to generate the complex for integration; the DNA sites important for assembly are simply not in the right place.

Phage X Excision Requires a New DN A-Bending Protein

How does X. excise? An additional architectural protein, this one phage-encoded, is essential for excisive recombination. This protein, called Xis (for excise), binds to specific DNA sequences and introduces bends in the DNA. In this manner, Xis is similar in function to IHF. Xis recognizes two sequence motifs present in one arm of afffl (and also present in attP— marked X, and X2 in Figure 11-9). Binding these sites introduces a large bend (> 140") and together, Xis, Mnt, and IHF stimulate excision by assembling an active protein-DNA complex at attR. This complex then interacts productively with proteins assembled at altL and recombination occurs.

In addition to Stimulating excision (recombination between attL and attR), DNA binding by Xis also inhibits integration (recombination between attP and offfi). The DNA structure created upon Xis binding to nttP is incompatible with proper assembly of Mnt and IHF at this site. Xis is a phage-encoded protein and is only made when the phage is triggered to enter lytic growth. Xis expression is described in detail in Chapter 10. Its dual action as a stimulatory cofaetor for excision and an inhibitor of integration ensures thai the phage genome will be free, and remain free, from the hosl chromosome when Xis is present.

Biological Roles of Site-Specific RFtcamhinntiori 305

The Hiri Recombinase Inverts a Segment of DNA Allowing Expression of Alternative Genes

The Salmonella Hm recombinase inverts a segment of the. bacterial chromosome to allow expression of two alternative sets of genes. Hin recombination is an example of a class of recombination reactions, relatively common in bacteria, known as programmed rearrangements. These reactions often function to "pre-adapt" a portion of a population to a sudden change in the environment. In the case of Hin inversion, recombination is used to help the bacteria evade the host immune system as we will now explain.

The genes that are controlled by the inversion process encode two filter native forms of flageliin {called the Hi and H2 forms]—the protein component of the flagellar filament. Flagella are on the surface of the bacteria and arc thus a common target for the immune system (Figure 11-11). By using Hin to switch between these alternative forms, at least some individuals in the bacterial population can avoid recognition of this surface structure by the immune system.

The chromosomal region inverted by Hin is about 1,000 bp and is flanked by specific recombination sites called hixL (on the left) and hixB (on the right) (Figure 11-12). These sequences are in inverted orientation with respect to one another. Hin. a serine recombinase, promotes inversion using the basic mechanism described above for this enzyme family. The inveriible segment carries the gene encoding Hin, as well as a promoter, which in one orientation is positioned to express the genes located outside the invertible segment directly adjacent to the hixH site. When the invertible segment is in the "on" orientation, these adjacent genes are expressed, whereas when the segment is flipped into the "off" orientation, the genes cannot be transcribed, because they lack a functional promoter.

The two genes under control of this "Hipping" promoter are fljB, which encodes the H2 flageliin, and fljA, which encodes a transcriptional repressor of the gene for the Hi flageliin. The HI fiagellm gene is located at a distant site. Thus, in the "on" orientation, H2 flageliin and the Hi repressor are expressed. These cells have exclusively HZ-type flagella on their surface. In the "off' orientation, however,

FIGURE 11-11 Micrograph of bacteria {Salmonella) showing flagella. Tbe color enhanced scanning electron micrograph shows Salmonella typhimunum (red) invading cultured human cells. The hair-like protrusions on the bacteria are the fiagefc. (Source: Cour tesy of the Rocky Mountain Laboratories, NIAID, NlH.)

FIGURE 11-11 Micrograph of bacteria {Salmonella) showing flagella. Tbe color enhanced scanning electron micrograph shows Salmonella typhimunum (red) invading cultured human cells. The hair-like protrusions on the bacteria are the fiagefc. (Source: Cour tesy of the Rocky Mountain Laboratories, NIAID, NlH.)

FIGURE 11-12 DNA inversion by the Hin recoffibmase of Salmonella. Inversion of the UNA segment between the/j« sites flips a promoter (P) tn give two alternative patterns of flageilin gene expression invertiblP segment hin l—■

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