A.  Introduction: 

      1. Homologous recombination vs. site-specific recombination:

            Homologous recombination occurs between DNA with extensive sequence homology anywhere within the homology. Site-specific recombination occurs between DNA with no extensive homology (although very short regions may be critical) only at special sites. The protein machinery for the two types of recombination differs too.  Strand exchange during site-specific recombination occurs by precise break/join events and does not involve any DNA loss or DNA resynthesis.

      2. Physical consequences of site-specific recombination events:

            a. Intermolecular reaction of circle + circle = integration

            b. Intramolecular reaction between two sites on circle with direct orientation = excision or resolution.

            c. Intramolecular reaction between two sites on circle with inverted orientation = inversion ("flipping").

      3.  Biological consequences of site-specific recombination events

            a.  Prophage integration/excision systems. Temperate bacteriophage establish lysogeny by integration of its genome (in a repressed state) into a special site on the bacterial chromosome by recombination at the attachment (or att) site.  Bacteriophage lambda integration excision is the best-characterized site-specific recombination system.  Other phage which integrate/excise include: P2, j80, P22 and 186. 

            b. Resolution/excision systems. 

                  1. Transposon cointegrate resolution.  Some transposons move by a replicative process that results in a "cointegrate" intermediate containing two copies of the transposon.  Site-specific recombination then occurs between the two copies of the transposons.  (More about this when we talk about transposition.) Transposons of this type include Tn3, gamma-delta, Tn21, Tn501 and Tn1721. 

                  2. Replicon dimer resolution. Homologous recombination between circular chromosomes or plasmids yields dimers, trimers, tetramers, etc. These larger structures do not segregate as well as the starting monomer plasmid. Therefore, some plasmids encode site-specific recombination systems to produce monomers from multimers.  Examples are the cer system of ColE1 plasmid and the cre/lox system of bacteriophage P1 which, in the lysogenic state, replicates as a plasmid. E. coli has a site-specific resolution site near the terminus of replication called “dif” at which the XerCD proteins act to resolve chromosome dimers to monomers before cell division. 

                  3. Developmental excision.  Bacillus subtilis undergoes a site-specific recombination event during sporulation to assemble a transcription factor specific for sporulation functions. The development of Anabena, a cyanobacterium, into the nitrogen-fixing heterocyst form involves two intramolecular site-specific recombination events to activate nitrogen fixation genes. 

            c. Inversion systems. 

                  1. Antigen switching/host range.  Site-specific inversion results in the expression of one of two forms.  Important mechanism for the generation of diversity within a population. Strategy is to avoid a mounting immune response against a particular antigen (in the case of Hin and Fim) or to prevent depletion of host, in case of Gin, Cin.  Inversion is reversible.  Examples: Hin: switching of Salmonella flagella antigen forms (stands for H inversion, H being flagellar genes).  Fim: switching of pilin antigens ("fimbriae") of E. coli. Gin, Cin: switching of host range of bacteriophage Mu and P1, respectively. Pin: function unknown, cryptic e14 prophage in E. coli. The Hin, Gin, Pin and Cin invertase proteins are highly homologous in amino acid sequence. 

                  2. FLP: The 2-micron plasmid of the yeast Saccharomyces cerevisiae inverts a segment.  This changes the relative orientation of the replication forks, promoting the amplification of the plasmid to high copy number. 

2.  The lambda Int system. 

      a. attP (the phage site) + attB (the bacterial site) results in the integration of lambda into the E. coli chromosome.  Integrated lambda DNA is flanked by two hybrid att sites:  attL (left) and attR (right)--each is derived from one half of attP and attB.  This process requires the lambda Int protein and the bacterial protein  IHF ("integration host factor"). IHF is a heterodimer of two E. coli proteins. 

      b. attL + attR results in the excision of lambda (restoring attP and attB) during the induction of lambda prophage. This process requires Int, IHF, and lambda Xis ( for "excise") proteins. E. coli Fis protein stimulates the excision but is not required absolutely. 

      c.  All att sites consist of a homologous 15 bp"core" region where the exchange takes place.  Flanking the core are two Int binding sites in inverted orientation.  The attP site is large, having 235 bp with complex series of binding sites for Xis, Int, IHF and Fis on either side of the core.  The attB is much simpler at 30 bp in size and consists only of the core and the two core Int binding sites. 

3. Mechanism of site-specific recombination integration: lambda Int. 

      a. The intasome. Specific proteins bind to the attP site forming a highly ordered nucleoprotein structure, wrapping 230 or so bp of DNA around a protein complex.  IHF induces sharp bend in the DNA of 140° when it binds  This bending is thought to be important for assembling the intasome complex. Int binds DNA both at the "core" region and at the two "arm" regions. These two modes of binding are different: different DNA sequences are recognized and the sites do not compete with each other. This bivalent (literally "double-strength") nature of Int binding is thought to allow subsequent interaction of the intasome and attB.  Binding is facilitated by superhelicity of attP DNA.  There are both cooperative and competitive interactions between binding proteins. These are thought be important for regulating and determining the directionality of the integration vs. the excision reaction. 

      b.  Synapsis. This part of the recombination process where the two molecules come together is not completely understood. The attB DNA is captured by the intasome complex.  Core homology is not required and synapsis must therefore be facilitated by protein interactions. 

      c.  Strand exchange.  

            1. Nicking occurs on each strand producing a 7 bp staggered cut.  The DNA is broken with the region of sequence identity of attP and attB. 

            2. No ATP or high-energy cofactor is required. The energy for the reaction derives from the cleavage of the phosphodiester bond of DNA, which is conserved via the formation of a transient enzyme-P-DNA linkage. DNA is joined via a 3' phosphate to a tyrosine residue of Int. 

            3. The phosphate is then transferred to the analogous strand of the second parental molecule, generating recombinant molecules with no nicks or gaps.  (This contrasts with homologous recombination where some post-recombination DNA repair synthesis and subsequent DNA ligation must occur. )  

            4.  Evidence suggests that the two strands are exchanged sequentially. That is, first the leftward strands are exchanged, then the rightward strands are exchanged. The first strand exchange, which results in the formation of a Holliday junction (crossed-strand structure), can occur even when there is no homology in the core regions between the two parental molecules. The first strand exchange is a reversible reaction, the second is not. 

4.  Regulation and directionality of lambda integration/excision. 

      a. Although you may think that excision is formally the reverse reaction of integration, the two recombination reactions are different mechanistically. Excision is an intramolecular reaction; integration an intermolecular recombination reaction. attL and attR are different in size and binding properties than attP and attB, because of the rearrangement of sequences.  The directionality of integration vs. excision is determined by the relative levels of the binding proteins in a complex way. Xis inhibits integration and promotes excision.  Although IHF is required for excision, binding of IHF to second site inhibits excision; Fis stimulates excision and has no effect on integration.

      b. These IHF and Fis effects allow directionality to be determined by growth phase of the host cell: IHF is high in stationary phase, Fis is high in log phase. The strategy here is that if the phage will be unable to replicate due to lack of growth nutrients delay excision (stationary phase: IHF high = inhibition of excision, Fis low = inhibition of excision).

      c. Under high Int/Xis conditions, these IHF/Fis effects are overridden.  Cooperative binding of these proteins allows more efficient excision.  High Int/Xis levels might be expected after induction of the SOS response, meaning strategically that if the cell is in trouble, lambda should get out while it can no matter what the growth phase.  Conversely, low Int/Xis can result from co-infection with other lambda  phage.  The strategy in this case is to not excise when competitors for replication are present, so the system become more sensitive to growth phase of the cell.

      d. In summary, the complex competitive and cooperative binding at the att sites allows the direction of phage recombination reactions to be adjusted to both host physiology and competing bacteriophage.

5. Use of site-specific recombination in genetic analysis

      a. High efficiency integration: The cre/lox system of bacteriophage P1 is being used as a simple integration system since the lox site is relatively small and recombination requires only the Cre protein and no other accessory factors. Circular segments of DNA containing a lox site can be transfected into cells and efficiently integrated at a resident lox site in the chromosome. 

      b.  Conditional expression. It is difficult to analyze the function of essential genes. In higher eukaryotes, if a gene is essential for early development, effects on later processes cannot be ascertained. Site specific recombination can be used to delete a function at a particular stage by flanking the gene by direct lox sites and by inducing expression of Cre with a regulatable promoter such as a heat-shock promoter. Cre will catalyze excision and deletion of the gene and the phenotype in the resultant organism can be assayed.

      c.  Mitotic recombination. A heterozygote for a particular mutation can be made and mitotic recombination that results in a homozygous mutant state can be induced in a subset of cells of the organism. A site-specific recombination site is placed centromere-proximal to the heterozygosity on the homologous chromosomes and expression of the site-specific recombination protein induced. Recombination between homologs will result in homozygous wild-type and homozygous mutant sectors. FLP and FRT (protein and sites of the site-specific recombination system of yeast 2 micron circle plasmid) have been used in this way in Drosophila.