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Evidence, largely from Irish pedigrees, indicates that a susceptibility locus for psoriasis maps to chromosome 4q. The gene encoding interferon regulatory factor-2 (IRF-2) maps to this region, and, as mice lacking IRF-2 exhibit a dermatologic phenotype resembling many aspects of human psoriasis, IRF-2 represents an attractive positional candidate. We set out to establish whether variation in IRF-2 sequence or expression was related to the development of psoriasis. First, the promoter, coding, and adjacent untranslated regions of IRF-2 were screened in individuals from 4q-linked families. Neither variant identified (IVS1A+29A/G; IVS3+729T/C), however, had functional credentials or statistical evidence supporting a susceptibility role. Second, in 62 Irish parent-offspring trios ascertained for psoriasis, we conducted a linkage-disequilibrium screen of the IRF2 region using a dense microsatellite map (covering 1.5 Mb from D4S1554 to D4S1540). Though there was nominal association for D4S1554/D4S2348 haplotypes (p=0.03) with one haplotype showing particularly skewed transmission to psoriatic offspring (p=0.0002, uncorrected), these markers lie some distance (500 kb) from IRF-2. Finally, we found no abnormalities of IRF-2 protein expression or distribution in skin biopsies from psoriatic individuals. These diverse lines of inquiry allow us to exclude variation in IRF2 as responsible for the 4q-linkage signal previously identified in Irish pedigrees.

Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis., (Copyright 2001 Academic Press.)

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one-dimensional diffusion along the DNA, or through three-dimensional space via multiple dissociation/re-associations. To distinguish these routes, reactions of the ECO:RV endonuclease were studied on substrates with two ECO:RV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one-dimensional diffusion. The variation in processivity with inter-site spacing shows instead that protein moves from one site to another through three-dimensional space, by successive dissociation/re-associations, though each re-association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA-binding proteins are usually thought to find their target sites by one-dimensional pathways, three-dimensional routes may be more common than previously anticipated.

To determine how distant sites on supercoiled DNA communicate with each other, the mechanism of site-specific recombination by resolvase was analysed by using a rapid-reaction quench-flow device to study the kinetics of individual steps in the reaction pathway. Three sets of measurements revealed the rates for: (1) the initial binding of the protein to its target sites on the DNA; (2) the synapsis of the two DNA-protein complexes; (3) the overall process of recombination. The binding of the protein to the DNA was complete within 50 milliseconds while recombination required 500 seconds. Surprisingly, synapsis spanned this entire time range: some DNA molecules gave synaptic complexes within ten milliseconds after the initial binding, while others took over 100 seconds. The departure from exponential behaviour may be due to each molecule of DNA having to undergo different conformational fluctuations in order to juxtapose the recombinational sites. From polymer physics theory, the rate of synapsis ought to vary with either the size of the DNA molecule or the length of DNA between the recombinational sites, depending on the nature of the fluctuations, but plasmids of different sizes and with different spacings between the sites all gave the same rates for synapsis. This observation cannot be reconciled with current models for encounters of distant sites on supercoiled DNA. However, the superhelical axis in the DNA molecules used here will be branched at one or more positions and the encounters may arise from the motion of a single branch relative to the remainder of the chain. Alternatively, the non-exponential kinetics for synapsis may be due to multiple re-arrangements of non-productive complexes following DNA juxtaposition.

Understanding how nucleoprotein complexes interact with specific DNA sequences has come closer with structural and mechanistic studies of the interaction between the recombination enzyme resolvase and DNA.

In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.

Tn3 and gamma delta resolvases catalyse site-specific recombination at res sites from Tn3 but not at Tn21 res sites. Tn21 resolvase has no activity at Tn3 sites and acts only at Tn21 sites. In both Tn3 and Tn21, res had three binding sites for the cognate resolvases; the cross-over site, I; and the accessory sites II and III, from which the bound proteins may stabilize the synaptic complex by protein-protein interactions. In this study hybrid res sites were made by replacing either II or III in the Tn21 res site with the equivalent sequence from Tn3. Plasmids containing either a hybrid and a wild-type Tn21 res site, or two hybrid sites, were tested for recombination. Relative to the reaction with two wild-type sites, recombination by Tn21 resolvase was reduced by replacing II at one res site and it was reduced further by replacing II at both loci but, in both cases, Tn21 recombination was enhanced by Tn3 or gamma delta resolvases. Very few of the amino acid on the external surface of gamma delta resolvases are conserved in Tn21. Moreover, mutants of gamma delta resolvase with defective protein-protein interactions also enhanced Tn21 recombination at this hybrid site. The resolvase at II thus seems not to be involved in protein-protein interactions and its main role may be to bend the DNA to the required structure. The replacement of III in the Tn21 site with Tn3 sequence also reduced recombination by Tn21 resolvase, especially when both loci carried the alteration but, in contrast to before, Tn3 or gamma delta resolvases now inhibited the Tn21 reaction. Recombination thus seems to require identical proteins at I and III, perhaps to allow for protein-protein interactions.

The res sites, the loci for site-specific recombination by resolvase, contain three binding sites for the protein; the cross-over site, I, and accessory sites II and III. The role of DNA bending by resolvase was examined by replacing either II or III in the res site from Tn21 with the recognition sequence for a heterologous DNA-bending protein, CAP (the catabolite-gene activator protein from Escherichia coli). The CAP sequence was placed at either the same position as the target sequence for Tn21 resolvase or a different position along the DNA. The activity of Tn21 resolvase for recombination between each hybrid and a wild-type res site was measured in the presence of CAP and cyclic AMP. When III was substituted, CAP inhibited Tn21 recombination, except when the CAP sequence was placed sufficiently far away from site II to allow resolvase to bind non-specifically to the DNA between II and the CAP site. With the substitutions at II, the extent of Tn21 recombination in the presence of CAP varied with the position of the CAP sequence: more recombination was observed when it superimposed the target sequence for resolvase than when it was displaced by five base-pairs. Efficient recombination by Tn21 resolvase thus seems to demand the cognate protein at site III in res, presumably for protein-protein interactions in the synaptic complex, while the function of resolvase at site II can be fulfilled, at least in part, by a heterologous DNA bend.

The EcoRV restriction endonuclease cleaves DNA not only at its recognition sequence but also at most other sequences that differ from the recognition site by one base pair. Compared to the reaction at the recognition site, the reactions at noncognate sites are slow but 1 out of the 12 noncognate sites on the plasmid pAT153 is cleaved more than 50 times faster than any other. The increase in the reaction rate at the preferred noncognate site, relative to other sites, was caused by the DNA sequences in the 4 base pairs from either side of the site. For enhanced activity by EcoRV, particular bases were needed immediately adjacent to the site, inside the DNA-protein complex. At these loci, the protein interacts with the phosphate groups in the DNA and the flanking sequence may control the activity of the enzyme by determining the conformation of the DNA, thus aligning the phosphate contacts. But the preferential cleavage also depended on sequences further away from the site, at loci outside the complex. At external positions, beyond the reach of the protein, the EcoRV enzyme required flanking sequences that give rise to flexibility in DNA conformation. These may facilitate the distortion of the DNA required for catalysis by EcoRV.

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.

Substitution of amino acids within the section of Tn21 resolvase that corresponds to a helix-turn-helix structure, with the equivalent residues from Tn3 resolvase, yields proteins that retain the ability to mediate recombination between res sites from Tn21. These proteins had no recombinational activity on res sites from Tn3, even when the complete recognition helix had been exchanged. In this study, the binding of these mutants of Tn21 resolvase to DNA fragments containing res from either Tn21 or Tn3 was analysed by DNase I footprinting and by gel retardation. With DNA containing res from Tn21, the mutants bound to all three of the binding sites for resolvase (I, II, and III) but with a lower affinity than wild-type Tn21 resolvase. No complexes were detected between Tn3 resolvase and Tn21 DNA. With DNA containing res from Tn3, both the mutants and wild-type Tn21 resolvase bound to sites II and III, forming similar complexes to those with Tn3 resolvase: some of the mutants had higher affinities for these two sites on Tn3 DNA than on Tn21 DNA. In contrast, at site I in res from Tn3 (the location of the recombinational cross-over), the derivatives of Tn21 resolvase formed aberrant complexes whose structures differed radically from that with Tn3 resolvase. Alterations in the amino acid sequence of resolvase, within the helix-turn-helix region, therefore modulate the affinity of the protein for its target sequence in the DNA, but the specificity of resolvase for recombination at its cognate res sites is determined by the resultant organization of the DNA-protein complex.