The use of local anaesthesia in haemorrhoidal banding: a randomized controlled trial.

AIM: Rubber band ligation is a common office procedure for the treatment of symptomatic haemorrhoids. It can be associated with pain and vasovagal symptoms. The effect of local anaesthetic use during banding was studied. METHOD: A single-blinded randomized controlled trial was carried out in the colorectal outpatient clinic. Patients presenting with symptomatic haemorrhoids suitable for banding were prospectively recruited and randomized to undergo the procedure with local anaesthetic or without (control). Submucosal bupivacaine was injected immediately after banding just proximal to the site. Vasovagal symptoms were assessed at the time of banding and pain scores (visual analogue scale) were recorded at the conclusion of the procedure, after 15 min, and on leaving the clinic. RESULTS: Seventy-two patients (40 local anaesthetic injection, group 1; 32 no injection, group 2) were recruited. The mean ages were 50 and 54 years respectively, the median duration of symptoms was 12 months in each group and the median number of haemorrhoids banded was three in each group. The mean pain score on leaving the clinic was 2.6 (95% CI 2.1, 3.1) in group 1 and 4.1 (95% CI 3.3, 5.0) (P = 0.04) in group 2. There were no complications related to local anaesthetic use. No significant difference in vasovagal symptoms was found (P = 0.832). CONCLUSION: Local anaesthetic injection at the time of banding is simple and safe. It may reduce patient discomfort following banding of haemorrhoids.

Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle).

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.

Nucleoside triphosphate-dependent restriction enzymes.

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.

Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems.

Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.

Restriction enzymes in cells, not eppendorfs.

Restriction enzymes are essential reagents to molecular biologists, but their relevance to bacterial populations is less obvious. Most bacteria encode restriction and modification systems and these are commonly considered to be a barrier to phage infection. Current evidence also supports a more general role for them in genetic recombination.

Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda.

The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.

Sequence diversity among related genes for recognition of specific targets in DNA molecules.

Escherichia coli strains K12 and B, and a new strain designated D, each encode a characteristic restriction and modification enzyme. These enzymes (EcoK, EcoB and presumably EcoD) comprise three subunits of which one, that encoded by the so-called specificity gene (hsdS), is responsible for recognition of the DNA sequence specific to that system. The other two subunits, encoded by hsdR and hsdM, are interchangeable between systems, and the available molecular evidence suggests that the hsdR and hsdM genes are highly conserved. The DNA sequence of a segment of the hsd region that includes the hsdS gene has been determined for each of the three strains. The hsdS gene varies in length from 1335 to 1425 base-pairs and the only regions showing obvious homology, one of about 100 base-pairs and a second of about 250 base-pairs, are highly conserved. The remainder of each hsd S gene shares little, or no, homology with either of the other related specificity genes. Thus, the specificity subunits, though components of a family of closely related enzymes with very similar functions, have remarkably dissimilar primary structure.

Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity.

We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype.

Mutations that confer de novo activity upon a maintenance methyltransferase.

DNA methyltransferases are not only sequence specific in their action, but they also differentiate between the alternative methylation states of a target site. Some methyltransferases are equally active on either unmethylated or hemimethylated DNA and consequently function as de novo methyltransferases. Others are specific for hemimethylated target sequences, consistent with the postulated role of a maintenance methyltransferase in perpetuating a pattern of DNA modification. The molecular basis for the difference between de novo and maintenance methyltransferase activity is unknown, yet fundamental to cellular activities that are affected by different methylation states of the genome. The methyltransferase activity of the type I restriction and modification system, EcoK, is the only known prokaryotic methyltransferase shown to be specific for hemimethylated target sequences. We have isolated mutants of Escherichia coli K-12 which are able to modify unmethylated target sequences efficiently in a manner indicative of de novo methyltransferase activity. Consistent with this change in specificity, some mutations shift the balance between DNA restriction and modification as if both activities now compete at unmethylated targets. Two genes encode the methyltransferase and all the mutations are loosely clustered within one of them.

Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme.

The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. We have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed. EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs. The protection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPgammaS are substituted for ATP. It is proposed that the DNA binding surface of EcoKI comprises three regions: a "core" region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit. The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint.

The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI.

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli.

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.

DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine.

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes.

We have identified the recognition sequence for the Citrobacter freundii restriction endonuclease CfrA, a member of the A-family of type I R-M enzymes. This bipartite target sequence differs in both its components from those of other type I enzymes. We determined the nucleotide sequence of its specificity gene (hsdS) and a comparison of this with its relative EcoA identifies two extensive variable regions, an organization analogous to that found in the K-family of type I R-M enzymes. The specificity polypeptides of the A-family, unlike those of K, have an N-terminal conserved region, and this includes a sequence repeated within the central conserved region. A second repeat sequence, identified at the amino acid level, coincides with the only sequence similarity common to all type I S polypeptides. Sequences immediately downstream from the hsdS genes of EcoA, CfrA, EcoK, B and D are almost identical, consistent with an allelic chromosomal location.

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.

The diversity of alleles at the hsd locus in natural populations of Escherichia coli.

In enteric bacteria three discrete families of type I restriction and modification systems (IA, IB and ID) are encoded by alleles of the serB-linked hsd locus. Probes specific for each of the three families were used to monitor the distribution of related systems in 37 of the 72 wild-type Escherichia coli strains comprising the ECOR collection. All 25 members of group A in this collection were screened; 12 were probe-positive, nine have hsd genes in the IA family, two in the IB and one in the ID. Twelve strains, representing all groups other than A, were screened; five were probe-positive, one has hsd genes in the IA family, one in the IB and three in the ID. The type ID genes are the first representatives of this family in E. coli, the probe-negative strains could have alternative families of hsd genes. The type IA and IB systems added at least five new specificities to the five already identified in natural isolates of E. coli. The distribution of alleles is inconsistent with the dendrogram of the bacterial strains derived from other criteria. This discrepancy and the dissimilar coding sequences of allelic hsd genes both imply lateral transfer of hsd genes.

Modification enhancement and restriction alleviation by bacteriophage lambda.

The activity of EcoKI, and related restriction and modification (R-M) systems, is modulated by the bacteriophage lambda ral gene product. We have identified the coding sequence for an analogous function in the Rac prophage of E. coli K-12.

Mutational analysis of conserved amino-acid motifs in EcoKI adenine methyltransferase.

The EcoKI methyltransferase (M.EcoKI, MTase) contains the amino acid (aa) sequences AAGTA and NPPF believed to represent the two sequences that are strongly conserved in adenine MTases [Klimasauskas et al., Nucleic Acids Res. 17 (1989) 9823-9831]. We have analysed a mutation in the first sequence that abolishes cofactor binding and enzyme activity, and mutations in the second sequence that reduce or abolish activity without affecting cofactor and DNA binding.