Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu

G inversion in bacteriophage Mu requires the product of the DNA invertase gene gin and an Escherichia coli host factor termed FIS (factor for inversion stimulation). A recombination substrate must contain two recombination sites, arranged as inverted repeats, and a recombinational enhancer sequence termed sis. FIS has been purified to homogeneity. The purified protein has a relative molecular weight of 12,000 when analyzed under denaturing conditions. The intact protein behaves as a dimer of relative molecular weight 25,000 in gel filtration analysis. The purified protein does not possess any recombinogenic activity when assayed in the absence of the DNA-invertase Gin. In the presence of purified Gin FIS is the only additional protein required for efficient inversion. By performing gel retention assays, we show that FIS is a DNA-binding protein, which specifically binds to DNA fragments containing the recombinational enhancer sis.     

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.     

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.     

The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9.

The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site-specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine. Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed.     

Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants.

The Gin recombination system of phage Mu mediates inversion of the DNA sequence between two sites (gix). In addition to Gin protein and gix sites, recombination requires an enhancer bound by the host factor FIS. We analyzed mutants of Gin that function in the absence of the enhancer and FIS and mediate deletion and intermolecular fusion in addition to inversion. The linking number changes caused by inversion imply that mutant Gin alone can form the same synaptic complex and can use the same strand exchange mechanism as the complete wild-type system. However, the linking number changes also reveal that unlike wild-type Gin, mutant Gin can recombine through more than one synaptic complex and can relax DNA in the absence of synapsis. This expanded repertoire allows mutant Gin to mediate DNA rearrangements not performed by wild-type Gin. Because mutant Gin, but not wild-type Gin, unwinds gix site DNA upon binding, we postulate that FIS and the enhancer function with (-) supercoiling to promote this unwinding with wild-type Gin. The analysis of the topological changes during DNA fusion shows that both the parallel gix site configuration and the right-handed rotation of the sites during exchange of wild-type Gin are a result of the (-) supercoiling of the substrate and the number of entrapped supercoils in the synaptic complex.     

Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

Gin mutants that can be suppressed by a Fis-independent mutation.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.     

Isolation and Genetic Analysis of Extragenic Suppressors of the Hyper-Deletion Phenotype of the Saccharomyces Cerevisiae Hpr1δ Mutation

The HPR1 gene of Saccharomyces cerevisae is involved in maintaining low levels of deletions between DNA repeats. To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S. cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1δ mutation. The mutations defined five different genes called HRS for hyper-recombination suppression. They suppress the hyper-deletion phenotype of hpr1δ strains for three direct repeat systems tested. The mutations eliminated the hyper-deletion phenotype of hpr1δ strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1). None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions. Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1δ mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1δ and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1δ strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated. We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.     

Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants

The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques. The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination. HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis. As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.     

Interaction of Polycomb-group proteins controlling flowering in Arabidopsis

In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.     

Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats

Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinucleotide repeats, at unique sequences, or for TNR contractions in srs2 mutants were not altered. Srs2 is known to suppress inappropriate genetic recombination; however, the TNR expansion phenotype of srs2 mutants was largely independent of RAD51 and RAD52. Instead, Srs2 mainly functioned with DNA polymerase delta to block expansions. The helicase activity of Srs2 was important, because a point mutant lacking ATPase function was defective in blocking expansions. Purified Srs2 was substantially better than bacterial UvrD helicase at in vitro unwinding of a DNA substrate that mimicked a TNR hairpin. Disruption of the related helicase gene SGS1 did not lead to excess expansions, nor did wild-type SGS1 suppress the expansion phenotype of an srs2 strain. We conclude that Srs2 selectively blocks triplet repeat expansions through its helicase activity and primarily in conjunction with polymerase delta.     

Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin

An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold. We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro. DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix. FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination. FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion. On the basis of these data, we discuss the possibility of additional functions for FIS in E. coli.     

Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

Cin is a member of the hin family of complementing site-specific recombinases which regulate the alternate expression of genes by inverting DNA segments. Common characteristics of this family of recombination systems are the requirement for an enhancer-like element in cis and the specificity for inversely oriented recombination sites on the same DNA molecule. We have isolated two mutants of the Cin recombinase which will efficiently recombine a substrate lacking the enhancer. In addition, these mutant proteins also catalyse efficient recombination between sites in direct orientation or on different DNA molecules. Both mutations are due to single amino acid substitutions at different positions in the protein and the two mutants have slightly different phenotypes. The finding that the loss of enhancer dependence is coupled to a change in topological specificity leads us to conclude that the enhancer determines the specificity of the system for DNA inversion.