The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved ...within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme
EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M
2S
1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single
EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.
Type I restriction enzymes bind to specific DNA sequences but subsequently translocate nonspecific DNA past the complex in a reaction coupled to ATP hydrolysis and cleave DNA at any barrier that can ...halt the translocation process. The restriction subunit of these enzymes, HsdR, contains a cluster of seven amino acid sequence motifs typical of heli-case superfamily II, that are believed to be relevant to the ATP-dependent DNA translocation. Alignment of all available HsdR sequences reveals an additional conserved region at the protein N-terminus with a consensus sequence reminiscent of the P-D…(D/E)-X-K catalytic motif of many type II restriction enzymes. To investigate the role of these conserved residues, we have produced mutants of the type IB restriction enzyme EcoAI. We have found that single alanine substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit abolished the enzyme's restriction activity but had no effect on its ATPase and DNA translocation activities, suggesting that these residues are part of the active site for DNA cleavage.
Type III restriction/modification systems recognize short non‐palindromic sequences, only one strand of which can be methylated. Replication of type III‐modified DNA produces completely unmethylated ...recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation‐specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site‐specific, but EcoP15I‐modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme‐to‐site ratio, in that site‐saturating enzyme levels elicit cleavage exclusively between the closest pair of head‐to‐head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I‐DNA complexes.
The use of surgical outcome in the comparative assessment of the quality of surgical care is predicted on the development of proper models that adjust for the severity of the preoperative risk ...factors of the patient. The National Veterans Administration Surgical Risk Study was designed to collect reliable, valid data about patient risk and outcome for major surgery in the Veterans Health Administration (VHA) and to report comparative risk-adjusted surgical morbidity and mortality rates for surgical services in VHA. This study describes the rationale and methods used in the Risk Study and reports on the frequency distribution of the data elements that will be used in the development of risk-adjusted reporting of surgical outcome.
This study was a prospective observational study in which dedicated nurses collected preoperative, intraoperative, and outcome data on patients undergoing noncardiac operations using general, spinal, and epidural anesthesia in 44 Veterans Administration Medical Centers. Outcome measures included all cause mortality within the 30 days after the index procedure and 21 major morbidities.
Eighty-three thousand nine hundred fifty-eight cases meeting inclusion criteria were entered in the study between October 1, 1991 and December 31, 1993. Ninety-seven percent of patients were men, with a mean age of 60.1 +/- 13.6 (standard deviation) years. The most common preoperative risk factors were smoking (40.7 percent) and hypertension (36.1 percent). Of the patients, 84.6 percent had one or more risk factors. The most common procedures were transurethral resection of the prostate gland (6.7 percent), total knee replacement (3.1 percent), thromboendarterectomy (2.4 percent), partial colectomy (2.2 percent), and total hip replacement (2 percent). The unadjusted mortality rate was 3.1 percent at 30 days. The most common postoperative morbidities were pneumonia (3.6 percent), urinary tract infection (3.5 percent), and failure to wean from the ventilator at 48 hours postoperatively (3.2 percent). Seventeen percent of the patients have one or more major complications.
The Veterans Health Administration has successfully implemented an outcome reporting system for major surgery that prospectively collects patient risk and outcome information reliably and validly. Risk adjustment models and comparative hospital-specific rates of risk-adjusted outcomes are currently being developed.
Type I restriction-modification systems bind to non-palindromic, bipartite recognition sequences. Although these enzymes methylate specific adenine residues within their recognition sequences, they ...cut DNA at sites up to several thousand base-pairs away. We have investigated the mechanism of how
EcoR124II, a type IC restriction-modification system, selects the cleavage site. Restriction studies with different DNA constructs revealed that circular DNA requires only one non-methylated recognition sequence to be cut, whereas linear DNA needs at least two such sites. Cleavage of linear DNA is independent of site orientation. Further investigations of the linear substrates revealed a mechanism whereby the double-strand break is introduced between two recognition sequences. We propose a model for the selection of restriction sites by type I enzymes where two
EcoR124II complexes bind to two recognition sequences. Lack of methylation at a site stimulates the enzyme to translocate DNA on both sides of the recognition sequence. Thus the two complexes approach each other and, at the point where they meet, they interact to introduce a double-strand break in the DNA.
The hsdS subunit of a type IC restriction‐modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half‐sites separated ...by a non‐specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half‐site. We have shown previously that the 5′ half of hsdS can code for a functional substitute of the full‐length hsdS. Here we demonstrate that the 3′ half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full‐length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5′ hsdS mutant, the 3′ mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co‐expression of the 5′ hsdS mutant and the 3′ hsdS mutant along with hsdM regenerates the wild‐type methylation specificity. Thus, there is a free assortment of subunits in the cell.
This paper presents the nucleotide sequence of the mod-res operon of phage P1, which encodes the two structural genes for the EcoP1 type III restriction and modification system. We have also ...sequenced the mod gene of the allelic EcoP15 system. The mod gene product is responsible for binding the system-specific DNA recognition sequences in both restriction and modification; it also catalyses the modification reaction. A comparison of the two mod gene product sequences shows that they have conserved amino and carboxyl ends but have completely different sequences in the middle of the molecules. Two alleles of the EcoP1 mod gene that are defective in modification but not in restriction were also sequenced. The mutations in both alleles lie within the non-conserved regions.
The promoter elements responsible for the expression of the regulatory nif genes rpoN, nifA1 and nifA2 of Rhodobacter capsulatus were mapped by exonuclease-III-mediated deletions and by primer ...extension analysis. The rpoN promoter maps 600 bp upstream of rpoN and has the characteristic features of a -24/-12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect -24/-12 promoter element. The spacing and/or the helical phasing of these two promotor elements was found to be important for promoter function. In addition, an UAS half-site may contribute to optimal promoter function. The rpoN UAS can partially substitute for the UAS of the nifE promoter. An open reading frame with homology to Klebsiella pneumoniae NIFU was identified between the rpoN promoter and rpoN and termed nifU2 since another nifU-like gene (nifU1) is located in a conventional nifUSVW operon in nif region A. Thus, rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with a nifU analogous gene from an rpoN-dependent promoter. Mapping of the promoter elements involved in the expression of nifA copy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC in R. capsulatus. The DNA region preceding the mapped 5' ends of the nifA transcripts displays much less homology. The distance between the distal and proximal elements is about 100 bp.
A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different ...Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
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