must recomfcine with the R:* segment of the bottom DNA molecule. Likewise, the Rl segment of the top molecule must recombine with the R4 segment of the bottom DNA molecule. Once this DNA "swap" has occurred, the 3'OH ends of each of the cleaved DNA strands can attack the recombinase-DNA bond in their new partner segment. As discussed above, this reaction liberates the recom-binase and covalently seals the DNA strands to generate the rearranged DNA product.

Tyrosine Recombinases Break and Rejoin One Pair of DNA Strands at a Time

In contrast to the serine recombinases, the tyrosine recombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two strands (Figure 11-7). Consider two DNA molecules with their recombination sites aligned. Here also, four molecules of the recombinase are needed, one to cleave each of the four

top strand exchange

bottom strand exchange to finish recombination

FIGURE 11*7 Recombination by a tyrosine recombinase. Here the Kt and R5

subunits cleave the DNA in the first step (a); m the example shew, the protein becomes linked to the cut DMA by a 3' P tyrosine bond. Exchange of the first pair of strands occurs when the two 5' OH groups at tfie break sites each attack the protein DMA bond on the cither DIMA molecule (b). The second strand exchange occurs by the same mechanism, using the R2 and R4 subunits fc and d). (Source: From Craig N. et at 2002. Mobile DNA II, cdor plate I, chapter 2 © 2002 ASM Press.)

top strand exchange individual DNA strands. To start recombination, the subunits of recombinase bound to the left recombinase binding sites (marked as Rl and R3 in Figure ll-7a) each cleave the tup strand of the DNA molecule to which they are bound. This cleavage occurs at the first nucleotide of the crossover region. Next the right top strand from, the top (gray) DNA molecule and the right top strand from the bottom (red) DNA molecule "swap" partners. These two DNA strands are then joined, now in the recombined configurations. This "first strand" exchange reaction generates a branched UNA intermediate known as a Holliday junction (see Chapter 10) (Figure 11-7b).

Once the first strand exchange is complete, two more recombinase subunits (those marked R2 and R4) cleave the bottom strands of each DNA molecule (Figure lt-7r;). These strands again switch partners, and then are joined by the reversal of the cleavage reaction. This "second strand" exchange reaction "undoes" the Holliday junction, to yield the rearranged DNA products. In the next section we discuss bow these chemical steps occur in the context of the recombinase pro-tein-DNA complex.

Structures of Tyrosine Recombinases Bound to DNA Reveal the Mechanism of DNA Exchange

The mechanism of site-specific recombination is best understood for the tyrosine recombinases. Several structures of members of this protein class have been solved, and these structures reveal the recombinases "caught in the act" of recombination. One beautiful example is the structure of the Cre recombinase bound to two different configurations of the recombining DNA. Insights into the mechanisms derived from these structures are explained below. Cre is an enzyme encoded by phage PI, which functions to circularize the linear phage genome during infection. The recombination sites on the DNA, where Cre acts, are called lox sites. Cre-Iox is a simple example of recombination by the tyrosine recombinase family; only Cre protein and the lox sites are needed for complete recombination. Cre is also widely used as a tool in genetic engineering (see l3ox 11-1, Application of Site-Specific Recombination to Genetic Engineering).

The Cre-Jox structures reveal that recombination requires tour subunits of Cre, with each molecule bound to one binding site on the substrate DNA molecules [Figure 11-8). The conformation of the DNA is generally a square planner four-way junction (see the discussion of Holliday junctions in Chapter 10) with each "arm" of this junction bound by one subunit of Cre. Although at first glance the structures appear to have fourfold symmetry, this is not really the case. Cre exists in two distinct conformations with one pair of sub-units in conformation 1, shown in green, and the other pair in conformation 2, shown in purple (Figure ll-8b). Only in one of these conformations (the green subunits in the figure) can Cre cleave and rejoin DNA. Thus, only one pair of subunits is in the active conformation at a time. The pair of subunits in this active conformation switches as the reaction progresses. This switching is critical for controlling the progress of recombination and ensuring the sequential "one strand at a time" exchange mechanism.

Conservative Site-Specific Recombination

Was this article helpful?

0 0

Post a comment