In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny ...virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.
OBJECTIVE:Single-center studies have demonstrated that resection of cavity shave margins (CSM) halves the rate of positive margins and re-excision in breast cancer patients undergoing partial ...mastectomy (PM). We sought to determine if these findings were externally generalizable across practice settings.
METHODS:In this multicenter randomized controlled trial occurring in 9 centers across the United States, stage 0–III breast cancer patients undergoing PM were randomly assigned to either have resection of CSM (“shave” group) or not (“no shave” group). Randomization occurred intraoperatively, after the surgeon had completed their standard PM. Primary outcome measures were positive margin and re-excision rates.
RESULTS:Between July 28, 2016 and April 13, 2018, 400 patients were enrolled in this trial. Four patients (2 in each arm) did not meet inclusion criteria after randomization, leaving 396 patients for analysis196 in the “shave” group and 200 to the “no shave” group. Median patient age was 65 years (range; 29–94). Groups were well matched at baseline for demographic and clinicopathologic factors. Prior to randomization, positive margin rates were similar in the “shave” and “no shave” groups (76/196 (38.8%) vs. 72/200 (36.0%), respectively, P = 0.604). After randomization, those in the “shave” group were significantly less likely than those in the “no shave” group to have positive margins (19/196 (9.7%) vs. 72/200 (36.0%), P < 0.001), and to require re-excision or mastectomy for margin clearance (17/196 (8.7%) vs. 47/200 (23.5%), P < 0.001).
CONCLUSION:Resection of CSM significantly reduces positive margin and re-excision rates in patients undergoing PM.
Background
Breast-conserving surgery (BCS) is a mainstay for breast cancer management, and obtaining negative margins is critical. Some have advocated for the use of preoperative magnetic resonance ...imaging (MRI) in reducing positive margins after BCS. We sought to determine whether preoperative MRI was associated with reduced positive margins.
Patients and Methods
The SHAVE/SHAVE2 trials were multicenter trials in ten US centers with patients with stage 0–3 breast cancer undergoing BCS. Use of preoperative MRI was at the discretion of the surgeon. We evaluated whether or not preoperative MRI was associated with margin status prior to randomization regarding resection of cavity with shave margins.
Results
A total of 631 patients participated. Median age was 64 (range 29–94) years, with a median tumor size of 1.3 cm (range 0.1–9.3 cm). Patient factors included 26.1% of patients (165) had palpable tumors, and 6.5% (41) received neoadjuvant chemotherapy. Tumor factors were notable for invasive lobular histology in 7.0% (44) and extensive intraductal component (EIC) in 32.8% (207). A preoperative MRI was performed in 193 (30.6%) patients. Those who underwent preoperative MRI were less likely to have a positive margin (31.1% versus 38.8%), although this difference was not statistically significant (
p
= 0.073). On multivariate analysis, controlling for patient and tumor factors, utilization of preoperative MRI was not a significant factor in predicting margin status (
p
= 0.110). Rather, age (
p
= 0.032) and tumor size (
p
= 0.040) were the only factors associated with margin status.
Conclusion
These data suggest that preoperative MRI is not associated margin status; rather, patient age and tumor size are the associated factors.
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.
Summary
A type I restriction‐modification enzyme will bind to an unmethylated target sequence in DNA and, while still bound to the target, translocate DNA through the protein complex in both ...directions. DNA breakage occurs when two translocating complexes collide. However, if type I restriction‐modification systems bind to unmodified target sequences within the resident bacterial chromosome, as opposed to incoming ‘foreign’ DNA, their activity is curtailed; a process known as restriction alleviation (RA). We have identified two genes in Escherichia coli, rnhA and recG, mutations in which lead to the alleviation of restriction. Induction of RA in response to these mutations is consistent with the production of unmodified target sequences following DNA synthesis associated with both homologous recombination and R‐loop formation. This implies that a normal function of RA is to protect the bacterial chromosome when recombination generates unmodified products. For EcoKI, our experiments demonstrate the contribution of two pathways that serve to protect unmodified DNA in the bacterial chromosome: the primary pathway in which ClpXP degrades the restriction endonucleas and a mechanism dependent on the lar gene within Rac, a resident, defective prophage of E. coli K‐12. Previously, the potential of the second pathway has only been demonstrated when expression of lar has been elevated. Our data identify the effect of lar from the repressed prophage.
Experiments using phage lambda provided early insights into important molecular mechanisms, including genetic recombination and the control of gene expression. Before recombinant DNA technology, the ...use of lambda, most particularly lambda transducing phages, illustrated the importance of cloning bacterial genes, already providing some insight into how to use cloned genes to advantage. Subsequently, lambda made significant contributions to recombinant DNA technology, including the early generation of genomic and cDNA libraries. More recently, lambda genes associated with recombination have enabled techniques referred to as 'recombineering' to be developed. These techniques permit the refined manipulation, including mutation, of foreign genes in Escherichia coli and their subsequent return to the donor organism.
Summary
It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of ...one such bacterium (Escherichia coli K‐12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated. Thus, the resident chromosome is recognized as ‘self’ rather than ‘foreign’ even in the absence of modification. We now provide evidence that restriction alleviation may be a characteristic of Type I restriction–modification systems, and that it can be achieved by different mechanisms. Our experiments support disassembly of active endonuclease complexes as a potential mechanism. We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome.
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