eEurope 2002: Quality Criteria for Health related Websites

Background A number of organisations have begun to provide specific tools for searching, rating, and grading this information, while others have set up codes of conduct by which site providers can attest to their high quality services. The aim of such tools is to assist individuals to sift through the mountains of information available so as to be better able to discern valid and reliable messages from those which are misleading or inaccurate.
Introduction
Health related web sites are now amongst the most frequently accessed sites on the internet with current estimates indicating that there are now over 100,000 sites offering health related information [1]. As a result of the wealth of information available and its apparent popularity, a number of organisations have begun to provide specific tools for searching, rating, and grading this information, while others have set up codes of conduct by which site providers can attest to their high quality services. The aim of such tools is to assist individuals to sift through the mountains of information available so as to be better able to discern valid and reliable messages from those which are misleading or inaccurate.
Recognising that European citizens are avid consumers of health related information on the internet and recognising that they are already using the types of rating system described above, the European Council at Feira on June 19-20 2000 supported an initiative within eEurope 2002 to develop a core set of Quality Criteria for Health Related Websites.
Accordingly a series of meetings was held during 2001 which drew together key players from Government departments, International Organisations, non-governmental organisations and industry, to explore current practices and experiments in this field. Some sixty invited participants from all the Member States, Norway, Switzerland, and the United States of America took part in the kick-off meeting of June 7-8, 2001: they included delegates from industrial, medical, and patient interest groups, delegates from Member States' governments, and key invited speakers from the field of health information ethics. These individuals, and many others, also took part in the web-based consultation which was open from august to November 2001 [2].
The focus of the discussions was primarily on the reliability of health related websites as a potential vehicle for health related messages, rather than on the substance and content of the health messages themselves. The specific aim was to draw up a commonly agreed set of simple quality criteria on which Member States, as well as public and private bodies, may draw in the development of quality initiatives for health related websites. These criteria should be applied in addition to relevant Community law [3].
As a result of the meetings, as well as a web-based public consultation, a core set of quality criteria was established. The criteria may be used as a basis in the development of user guides, voluntary codes of conduct, trustmarks, accreditation systems, or any other initiative adopted by relevant parties, at European, national, regional or organisational level. By using a common set of criteria as a starting point, such initiatives can develop in a focused manner across the European Union.
The objectives for the criteria were defined as follows:
The quality criteria should address issues of both supplier and user education: one document that simultaneously tells suppliers how to comply with key quality criteria and educates users as to what they ought to expect from a good health website;
The quality criteria should address both passive information-giving sites as well as sites that allow for transactions between service or information providers and users (i.e. information, products and services).
The quality criteria should facilitate compliance with EU Directives, other current guidelines, and technical standards relevant to this area.
It should be noted that the objective was not to develop a method for the implementation of such criteria at a European level. Although some actors in the field have called for an EU trustmark for health related websites which would operate in a way similar to the CE marking of certain goods [4,5], such initiatives are not within the ambit of the eEurope2002 action. They may, however, be considered within future eEurope action plans and other European programmes.
Quality Criteria for Health Related Websites
Developed in widespread consultation with representatives of private and public eHealth websites and information providers, other industrial representatives, public officials, and representatives of government departments, international organisations, and non-governmental organisations.
These criteria should be applied in addition to relevant Community law
Transparency and Honesty
Transparency of provider of site - including name, physical address and electronic address of the person or organisation responsible for the site (see Article 5 and 6 Directive 2000/31/EC on Electronic Commerce).
Transparency of purpose and objective of the site
Target audience clearly defined (further detail on purpose, multiple audience could be defined at different levels).
Transparency of all sources of funding for site (grants, sponsors, advertisers, non-profit, voluntary assistance).
Authority
Clear statement of sources for all information provided and date of publication of source.
Name and credentials of all human/institutional providers of information put up on the site, including dates at which credentials were received.
Privacy and data protection
Privacy and data protection policy and system for the processing of personal data, including processing invisible to users, to be clearly defined in accordance with community Data Protection legislation (Directives 95/46/EC and 2002/58/EC).
Updating of information
Clear and regular updating of the site, with date of up-date clearly displayed for each page and/or item as relevant. Regular checking of relevance of information.
Accountability
Accountability- user feedback, and appropriate oversight responsibility (such as a named quality compliance officer for each site).
Responsible partnering - all efforts should be made to ensure that partnering or linking to other websites is undertaken only with trustworthy individuals and organisations who themselves comply with relevant codes of good practice.
Editorial policy - clear statement describing what procedure was used for selection of content.
Accessibility
Accessibility- attention to guidelines on physical accessibility as well as general findability, searchability, readability, usability, etc.
Relevant Community Law is listed in reference 3. Terms in italics are further discussed in the Glossary of Terms.
It should also be noted that while this Communication is addressed to the Member States of the European Union and private or public bodies operating in those States, due consideration should be given to the global nature of information disseminated through websites. Accordingly bodies adopting measures to implement the criteria should be aware of the fact that their information will be accessed by many individuals of different nations and cultures. In particular, attention should be paid to the fact that the developing world is a keen consumer of health information and that culturally specific content should be clearly identifiable as such.
Textbox 1 sets out the resulting quality criteria; the ensuing text then explores some of the ways in which they may be implemented. This illustrates what could be done at national or regional level to promote high quality, accessible health related information to the European citizen. The table may easily be detached from the present text to form a simple reminder of the key Quality Criteria for Health related Websites.
Tailoring the Criteria For Different Types of Health Related Content
The criteria set out above are designed to be applicable to the development and maintenance of a health related site irrespective of the type of information or audience to whom the information is targeted. However, one essential quality criterion is that a health-related web site should state clearly what is its target audience and that care should be taken to ensure that both the style and nature of the information, and its presentation, are appropriate for the chosen audience. A number of the respondents to the consultation on the draft criteria, which was conducted between August and October 2001 via the eEurope website, identified the need to address not only site development and maintenance, but also the specific quality issues particular to health related content [6].
When tailoring the content to a chosen audience, a number of factors should be borne in mind in addition to those set out above which should govern the construction of a site. These factors may be considered under the same broad headings as the general site development criteria:
Transparency of Health Related Content
Transparency of the health related objectives of the provider of the information, including the purpose and objective of content provision, should be clearly defined and stated.
Where advice or information on particular conditions, lifestyles or medications is given, funding from producers of products thereby implicitly or explicitly endorsed should be transparent to the site user.
Existing Community legislation already contains information and transparency requirements. For example Article 5 of Directive 2000/31/EC on electronic commerce concerns the general information to be provided by an Information Society Services provider; Article 6 of Directive 2000/31 which concerns additional information to be provided in the case of commercial communications which are part of or constitute an information society service and Article 10 of Directive 95/46/EC on the protection of individuals with regard to the processing of personal data and on the free movement of such data also applies.
Authority of Health Related Content Providers
Where a policy of using only accredited medical professionals to generate content is adopted, this should be clearly stated and adhered to.
Where a mixed group of content providers is used, (medical professionals, journalists, personal testimony, etc) the category of content provider of each item should be clearly identifiable.
Where scientific evidence is cited, the sources of such evidence should be easily identifiable to the user.
Where a medicinal product is recommended, EU legislation on Medicinal Product advertising should be adhered to, and any documents authorised by a regulatory authority should be made available to the site user.
Where advice is offered, the site provider should always include a reminder that internet based advice, whether personalised or not, cannot replace a face to face consultation with a healthcare practitioner.
Privacy and data protection of Health Data
Where any personal information is collected and further processed by the site user, including data processing invisible to the users, the requirements of Directive 95/46/EC on the protection of individuals with regard to the processing of personal data and on the free movement of such data, in particular article 8 on sensitive and health data, should be carefully assessed and full compliance assured.
Updating of Health Related Information
Where specific health related data are provided, the relevance of such content should be regularly verified.
Accountability for Health Related Content
Where specific health related user feedback is provided by the site, particularly where personalised medical advice is offered, every effort should be made to ensure that such advice is bona fide and that advisors are suitably qualified to offer advice.
Accessibility in Health Related Content
Where a particular type of audience is targeted (eg children), the presentation and content of information should be appropriate to the chosen target audience.
The use of a metadata labelling system may be used to make health data more findable. Such a system may also be used in conjunction with quality criteria to give higher ranking by search engines to those sites or pages labelled as complying with defined quality criteria.
Apply International or European standards, wherever possible, in order to facilitate notably the interoperability between different services and the cross-border provision of web based health services.
Implementation of the Quality Criteria For Health Related Websites
Issues for the European Community
The purpose of the eEurope 2002 action on Quality Criteria for health-related Websites was to encourage the adoption of a common set of basic quality criteria for such sites. The issue of whether and how these criteria might be implemented at European level was not within the terms of the action. The implicit assumption was that this was a matter to be addressed in Member States at national or regional level, making use of the wide range of private and not-for-profit organisations which are already operating systems for implementing quality criteria for health-related websites.
In view of the rapid increase in health-related websites in the European Union and the increase in the number of European Union citizens consulting such sites, it could be argued that there would be merit in the Community establishing its own system for implementing agreed quality criteria. Such a Community-sponsored system would however require considerable resources to set up and operate, and it is unclear that it would offer clear value added to the Member States. The Commission therefore considers that at the moment the difficulties inherent in a Community system would outweigh any possible advantages. Nevertheless, the issue of how and how effectively quality criteria are being implemented is of considerable significance at the European level. To ensure that European citizens have access to reliable health information on the Internet implies not only that there is a consensus on the necessary quality standards, but that those standards are satisfactorily implemented right across the European Union.
This does not mean that the same method of implementation should be used everywhere - indeed it must be doubtful that any particular mechanism would be appropriate in all circumstances and in all countries For example, in pharmaceuticals the Commission is considering ways of meeting the growing demand by patients to be able to access information directly about their medicines. The Commission has included proposals within the current review of EU pharmaceutical legislation, Review 2001, to take account of this growing demand. This has also been recently reinforced by the work of the High Level Group on Innovation and Provision of Medicines - G10 Medicines - which has covered this area in their reports.
However, whatever system adopted, there should be clarity about the mechanisms being used in the different Member States and the extent of the involvement of the national and regional health authorities. With the forthcoming enlargement of the European Union, this requirement for transparency becomes even greater.
Some Examples of Methods of implementing Quality Criteria
Simple Codes of Conduct
A number of organisations have adopted an approach similar to that described in this Communication, of setting up meetings and consultations between experts in order to establish by consensus a set of quality criteria. The eHealth Code of Ethics adopted in May 2000 by the Internet Health Coalition [7] is perhaps the best known of such `codes of conduct'.
The object of this and other similar codes is to offer a process of self-assessment by health site providers. However, a basic code of good conduct, or set of quality criteria will form the basis of all the approaches described below.
The way in which such codes are implemented varies. Where the code is adopted by an umbrella organisation, such as the Pharmaceutical Group of the European Union, then the organisation itself seeks to ensure that all members comply with the code. In other cases a code has been adopted for the purposes of in-house application only, as in the case of the American Medical Association. Although other organisations may cite the code, and claim to use it, the organisation developing the code makes no attempt to ensure that other parties are in fact implementing it.
The costs of the code of conduct approach are generally rather low, requiring only an initial outlay on meetings to draw up the code. However, the benefit of such codes can also be rather limited given the absence of effective enforcement mechanisms.
A code of conduct which addresses issues concerning the protection of personal could in itself form a Code of Conduct relevant to a specific area of practice as foreseen in Article 27 of Directive 95/46/EC on Data Protection. Any such draft community code, and amendments or extensions to existing Community codes, may be submitted to the Working Party established by Article 29 of Directive 95/46/EC on data protection. Similarly, a Code of Conduct which addresses the particular issues of electronic commerce in the health domain may be drafted in accordance with the framework foreseen in Article 16.1 of the Directive 2000/31/EC on Electronic Commerce.
Self Applied Code of Conduct or Quality Label
A next step in the implementation of a code of conduct can be characterised as the self applied quality label. In such a case a third party organisation develops a code of conduct and allows those who undertake to abide by the terms of the code to display a label, seal or logo which certifies compliance with the code.
The oldest, and perhaps best known, of such labels is the Health on the Net Foundation (HON) [8] label whose eight point set of quality criteria is currently used by more than 3000 internet sites worldwide. A site provider wishing to use the HON label has to make a formal application and a commitment to strictly observe all the HON code principles. Compliant sites identify themselves by the HON code hyperlink (or "active") seal displayed at a prominent location. The seal is termed `active' because clicking on it links the user to the HON site. Conformity with the HON code principles is verified by the team of checkers at HON. HON cannot prevent dishonest operators from simply cutting and pasting the HON code seal onto their Web sites in a bid to enhance their credibility. It does, however, conduct random checks on subscribers to ensure they remain compliant with the HON code. By way of additional policing, the Internet community is invited to report misuse of the label.
The costs of this system of applied labelling are not very high, requiring a relatively small team to process applications for use, maintain random checks of sites displaying the label and respond to any reports of misuse. The benefits may be significant in drawing to the attention of users the importance of the criteria inherent in the label. However, the benefits must be weighed against the requirement of the users of the sites to understand the nature of the label, and perhaps more importantly, to care about its aims and objectives.
User Guidance Tools
A further application of the code of good conduct takes the form of a user guidance tool. In this case compliance with a code is demonstrated not by a label, but by a link to a guidance tool which invites the user to check for him or herself if a site and its contents comply with pre-set criteria.
A typical such tool is displayed by the site as a logo on which the user may click to reveal a series of questions with which to interrogate the site so as to assess whether the information offered is trustworthy. Such tools may be specific to a particular type of information, such as DISCERN [9] which provides a brief questionnaire through which users gain a valid and reliable way of assessing the quality of written information on treatment choices for a health problem. Other tools seek to give guidance on the trustworthiness of any health-related information. An example of this is NETSCORING [10], which uses a questionnaire of 49 criteria falling into eight categories: credibility, content, links, design, interactivity, quantitative aspects, ethics, and accessibility. Yet other tools are targeted at particular categories of internet users. For example, the QUICK [11] tool seeks to provide children with a step-by-step guide to assessing health related information on the internet.
While such tools are frequently adopted for the guidance of users by national health portals (such as National Health Service DIRECT in England and Wales), they may also be used as site development tools by authors and publishers of information since they define the standards which users are entitled to expect.
The financial costs of the user guide are low, often not extending beyond the initial development costs. However, the burden of the use of this kind of tool falls on the internet user, mostly because of the time it takes to apply, which reduces the incentive to use it.
Filtering tools
Where a guidance tool is provided by a third party to a user to apply for him or herself, a filtering tool is applied to provide a searchable database of filtered and accredited information. Such filtering tools are often based on the gateway approach to organising access to Internet. The fundamentals of this approach are that Internet resources are selected for their quality and relevance to a particular target audience. They are then reviewed and resource descriptions created, which are stored, generally with the associated metadata, and generally in a structured database. The consequence of this effort is to improve the recall, and especially the precision, of Internet searches for a particular group of users.
An example of this type of tool is found in the OMNI site (Organising Medical Networked Information) [12] which provides a gateway to evaluated, quality Internet resources in health and medicine, aimed at students, researchers, academics and practitioners in the health and medical sciences.
The costs of such a filtering tool are relatively high in that a team of trained experts must be employed to search for, abstract and classify information on the internet in order that it may be entered into the database. The benefits of such a tool, for the initiated user, are also high since it provides a valuable shortcut to individual searches of the internet using non-specific search engines.
Third Party Quality and Accreditation Labels
The most advanced, and also most costly, of the mechanisms available for implementing quality criteria for health related websites, is the third party accreditation system. A third party issues a label to certify the compliance of the site with the criteria of evaluation.
A range of implementations fall into this category, from lower cost intra-organisation bodies for quality certification, acting in a similar to the notified bodies used in CE marking, to high cost external independent assessors who perform audits and grant accreditation.
At present no third party accreditation bodies are fully operational in Europe, although two noteworthy pilots are running in MEDCERTAIN (a demonstration project of the European Union "Safer Internet Action Plan") and TNO QMIC, a pilot study of the Netherlands Organisation for Applied Scientific Research.
In the case of MEDCERTAIN [13] a series of levels of accreditation are envisaged, starting with a self-certification label in which the provider of the site uses the MEDCERTAIN metalabelling system which incorporates a machine read language to describe and evaluate health information on the Internet. These labels are then in turn used to place a given item of site correctly within a gateway system, such as the OMNI system described above. The next level envisaged by MEDCERTAIN is one in which non-medical experts personally check the site for compliance with the level 1 tagging and also against the agreed set of quality criteria. The highest level involves medical assessment of the content and a rating of the content by relevant healthcare professionals.
The QMIC [14] system, on the other hand, envisages a system similar to the ISO 9000:2000 standard. The QMIC system is based on a complex set of standards drawn up by the third party (TNO in this case) but implemented by the site provider through an internal `quality certification body' who is in turn regularly assessed by the third party organisation to ensure that it is performing its function of internal quality assurance properly. The site, once duly assessed by the internal notified body is then admitted to a portal maintained by the third party who undertakes to ensure that the sites linked into the portal are applying the internal quality assurance system with due care.
Purposes of Implementation of Quality Criteria For Health Related Websites
The general purpose of any quality initiative, whatever method of implementation is chosen, must be the protection of the consumer. However, in some cases that general purpose may be best achieved through educating the user of the service while in other cases the provider of the service will be the target of the quality initiative. In order to assist in the selection of an appropriate implementation method, the targeted purposes of the various methods are examined in more detail below
Educating Users
In their daily lives as consumers of information delivered via the traditional media, most people learn to use a wide range of assessment tools: judging the nature of the outlet providing the information (a general or specialist bookshop or a work exclusively available from the author), the look and feel of the publication as a whole (a magazine with several contributions or a one page pamphlet). In addition, most people know whom to contact for further information (librarian, bookshop assistant, publisher).
In the world of internet content, however, it is less evident what are the relevant indicators of quality. It is for this reason that quality marks and user guides have proliferated, namely to educate the consumer and to provide a recognisable "quality" label which site creators may use to promote their sites. Accordingly, for such codes to be effective it is highly important that the public are informed about the existence of the Codes through public education campaigns.
Assisting searchers
The purpose of quality marks is not, however, simply to provide access to qualified information but also to assist the citizen in coping with the torrent of information which a search on a health related subject might produce: it has been said that "trying to get information form the internet is like drinking from a fire hose. You don't even know what the source of the water is" [15]. In order to try to manage the flow from the fire hydrant into a steady stream from a tap, some organisations have developed and applied tools for rating web sites in order that they may offer pre-selected and more easily searchable sources to their consumers (see for example OMNI or MEDCERTAIN).
Educating Site Providers
The problem is not only with the torrent of information, but also with the behaviour of its purveyors. Whilst it may take considerable effort to find an outlet for unusual or extreme ideas in the traditional media, virtually anyone with a modicum of computer skills and very little money can create their own website. The objective of many of the code of conduct initiatives is therefore to educate both the providers and consumers of information about the processes and good practices that a website should be able to demonstrate.
In order to educate not only the provider, but also the consumer of information a further set of actors have developed a wide range of user assessment tools. Such tools are usually in the form of on-line check lists which ask the consumer to check off types of information as they find them: statement of aim, explicit statement of source of information, explicit date of information, etc. Some may be rather short (HON), some quite detailed (NETSCORING); some are aimed at specific markets (DISCERN - for treatment choices) and some aimed at children (QUICK) to mention but a few.
Assuring Quality
Most of the organisations publishing and administering such codes operate on a simple selflabelling processes in which the site provider undertakes to follow the code and in return displays its "trustmark" relying on spot check and vigilant users to identify those who are not complying with the given code of conduct. While this may not be as effective as a fully policed trustmark system of the type we are used to seeing as regards, for example, electronic products, it nonetheless addresses a need in a reasonably effective manner.
Conclusions
The eEurope initiative was launched by the European Commission on 8th December 1999, with the adoption of the Communication `eEurope - An Information Society for all (COM (1999), 687 final, of 8.12.1999)'.
The "eEurope 2002 Action Plan - An Information Society For All", was adopted by the Commission on 14th June 2000, and politically endorsed by the European Council in Feira (Portugal) on 19-20 June 2000. It detailed the policy actions which are required to meet these objectives by 2002.
The eEurope 2005 Action Plan (COM (2002) 263 final, of 28.5.2002), was adopted by the Commission on 28 May 2002 and politically supported by the European Council in Sevilla (Spain) on 21 - 22nd June 2002. It, notably, set the objective for Europe to have, by 2005, " Modern online public services". To achieve this objective, one of the proposed actions is to promote e-health services. It also commits the Commission to monitor " actions taken by Member States to make health information as accessible as possible to citizens as well as initiatives to implement quality criteria for web sites".
In this respect, the e Europe 2005 Action Plan affirms that " it is critical that e-health content and services are developed efficiently, are available for all and health related web sites comply with established quality criteria".
With respect to the enlargement of the European Union it should also be noted that the eEurope+ Action Plan, which was adopted by the accession States to mirror the eEurope 2002 Action Plan, includes similar action on quality criteria for health related websites.
Accordingly it will also be important to monitor the activities undertaken pursuant to that Action Plan.
This Communication sets the scene for the implementation of a core set of quality criteria in Member States for health related web sites, within the context of the relevant existing Community legislation (as listed in footnote 3) and in accordance with the requirements of that legislation. The set of quality criteria is based upon a broad consensus among specialists in this field, health authorities, and prospective users. It is now to be expected that national and regional health authorities, relevant professional associations, and private medical website owners will:
implement the Quality Criteria for Health Related Websites in a manner appropriate to their website and consumers.
develop information campaigns to educate site developers and citizens about minimum quality standards for health related websites.
draw on the wide range of health information offered across the European Union and localise such information for the benefit of citizens (translation and cultural adaptation).
exchange information and experience at European level about how quality standards are being implemented.
Finally, within the context of the Information Society activities and as part of the implementation of the European Union public health programme, consideration will be given to the possibilities of developing and operating a joint action, with the plans drawn up under eEurope, to improve availability to the general public on the Internet of information on health matters, and considering the possibilities for establishing a system of recognizable Community seals of approval for Internet sites.

guidelines
privacy
quality assurance
internet/standards
ethics, professional
social control, formal
health care quality
health care/standards
commerce/standards
information management/standards
medical informatics/standards
quality control
informed consent


Restriction Endonucleases that Bridge and Excise Two Recognition Sites from DNA

Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.
Introduction
Restriction–modification (R-M) systems can be classified into four main groups on the basis of their genetic and polypeptide organisations, their modes of action and their co-factor requirements: Types I, II, III and IV.1,2 Most bacterial genomes encode multiple R-M systems: about 35% of the total are Type I systems, 40% Type II, 10% Type III and 15% Type IV.3 The restriction enzymes from the Type I and the Type III systems are ATP-dependent proteins that initiate DNA translocation at specific recognition sites and then cleave DNA some distance from the sites, usually upon collision of two complexes translocating from separate sites.4,5 Type I restriction enzymes normally require two recognition sites in cis to cleave DNA, though they can cleave circular DNA with one site, while the Type III nucleases almost always need two sites.6,7 The Type IV systems are GTP-dependent enzymes that restrict DNA that has been methylated at two or more sites.5,8 The endonucleases from the Type I, III and IV families thus constitute ∼60% of the restriction enzymes found in nature, and they all interact with two target sites before cleaving DNA.9
While the Type I and Type III systems feature multi-subunit proteins with both endonuclease and methyltransferase activities,4,5 Type II R-M systems generally comprise two separate proteins, one for each activity,10 though in some cases both functions are fused into a single polypeptide.11,12 Unlike the NTP-dependent systems, the Type II endonucleases cleave DNA at fixed positions in or near their recognition sites.3 Most (but not all)13 need Mg2+ as a cofactor,10,14 though some of the fused proteins additionally need the methyl donor S-adenosyl methionine (AdoMet) not only for methyltransferase but also for nuclease activity.11,12 The standard Type II restriction enzymes, such as EcoRV and BamHI, are homodimers that recognise palindromic sequences, typically 4–8 bp long, and cut both strands at specified positions within the sequence.14,15 They cleave DNA with multiple sites by means of separate reactions at each site.16–18
Of the ∼4000 Type II restriction enzymes identified to date3 many differ from the standard and have been classified into various sub-types.2 One tenet of this classification is their mode of action at multiple recognition sites. Numerous Type II enzymes, maybe 50% of the total,9,18 cannot cleave DNA without first interacting with two copies of their sites, either looping out the DNA between two sites in cis on the same chain of DNA, or bridging sites in trans on separate molecules of DNA.19–23 The enzymes that need two sites can be identified by comparing their activities on DNA substrates with one or two copies of the requisite sequence.24,25 Under most circumstances, an enzyme that binds two DNA loci has a higher affinity for sites in the same chain over sites in separate molecules,9 so will usually cleave two-site DNA more rapidly than one-site DNA.24 However, an enzyme that needs two sites but which has a high affinity for its site may have sufficient affinity for sites in trans to give its Vmax on a DNA with one site.25 Nevertheless, DNA–protein interactions are weakened at elevated ionic strengths so even if an enzyme gives equal rates on the one and two-site substrates at low ionic strength, it may cleave the two-site DNA faster than the one-site DNA at raised ionic strength.16,18,26 Alternatively, these enzymes can be identified from whether they can be activated to cleave plasmids with one target site by adding oligonucleotide duplexes that also carry the target sequence, as a result of interactions in trans between plasmid and oligoduplex.20,25,27
The Type II restriction enzymes that need two sites have been categorised into different sub-types on the basis of how they cleave DNA with two or more copies of their site.2 The Type IIE enzymes, such as EcoRII, NaeI and Sau3AI,27–29 have two (or more)30 dissimilar DNA-binding clefts: one has the catalytic functions for cleaving the recognition sequence but is inactive unless a second copy of the sequence is bound to an activator cleft. The Type IIF enzymes, such as SfiI, NgoMIV and SgrAI, function as tetramers with two identical DNA-binding clefts, each made from two subunits22,31 but they are virtually inactive unless both clefts are filled with cognate DNA.24,32 Consequently, while both Type IIE and IIF enzymes cleave two-site substrates more rapidly than one-site substrates, Type IIE enzymes cut two-site DNA initially at just one site and then, in a much slower reaction, the second site.33 In contrast, Type IIF enzymes act concertedly, to give as their initial product DNA cut at both sites.22,24,34,35
Type II restriction enzymes have also been categorised on the basis of the positions at which they cleave the DNA relative to their recognition sequences.2 While many Type II enzymes cleave within palindromic sites, the Type IIS nucleases recognise asymmetric sequences and cleave both strands at specified positions on one side of the site.3 For example, FokI cleaves DNA downstream of the sequence GGATG, 9 and 13 bases away in “top” and “bottom” strands, respectively. Its recognition site is thus usually noted as GGATG(9/13). Most Type IIS nucleases, including FokI, have very low activities against DNA with one cognate site, and become active only after interacting with two sites.36 However, they vary in their modes of action at two DNA sites: some cut just one strand at one site,13 others both strands at one site, like Type IIE enzymes,36–38 while further examples cut both strands at both sites, like Type IIF enzymes.39,40
Categorisation of R-M systems from their positions of DNA cleavage yields a further subset,2 the Type IIB enzymes (Table 1) exemplified by BcgI.41–45 The Type IIB systems have bipartite recognition sequences that are usually asymmetric though some, such as BplI and FalI, have palindromic sites (Table 1).3 Their recognition sites look like sites for Type I rather than Type II systems but the Type IIB systems belong unambiguously to the II family because their endonucleases cut DNA at specified rather than random positions. Even so, the similarities between the Type I and the IIB systems extend to their functional organisations.42,44,46–48 Like Type I enzymes,4 each IIB system features a multi-functional protein with both methyltransferase and endonuclease activities. BcgI contains two polypeptides, A and B, in a 2:1 ratio.42 The A subunit has the amino acid sequences for both methylation and cleavage activities but by itself has neither activity. The B subunit resembles a DNA specificity subunit from a Type I enzyme and is thus likely to direct the protein to its recognition site.44 Several other Type IIB enzymes contain two subunits akin to the A and B subunits of BcgI,49 though in BplI these are at a 1:1 rather than a 2:1 ratio.50 In contrast, some IIB enzymes carry the endonuclease, the methyltransferase and the DNA recognition functions in three separate blocks of amino acid sequence within a single polypeptide.46–48 The IIB systems all require AdoMet for DNA methylation and Mg2+ for DNA cleavage but, as is common among combined restriction–modification proteins, many also need the methylation cofactor for their nuclease activity (Table 1).43
The positions of DNA cleavage by Type IIB nucleases are, like Type IIS enzymes, fixed distances from their recognition sites, 7–15 bases away depending on the system in question (Table 1).3 While the Type IIS enzymes make a double-strand break on one side of the site, the Type IIB nucleases make double-strand breaks on both sides of their sites,41,46–50 so cutting a total of four phosphodiester bonds at each site. They thus release their intact recognition sites from the remainder of the DNA on a short fragment, 30–35 bp long depending on the enzyme (Table 1). Perhaps as a result of remaining bound to its 34 bp product, the BcgI nuclease functions stoichiometrically, completing just one turnover, rather than catalysing multiple turnovers.42–45 However, the complete cleavage of a plasmid with two BcgI sites required less of the BcgI protein than a plasmid with one site, so it was suggested that BcgI interacts with two copies of its target sequence before cutting DNA.45 Many restriction enzymes have to bridge two sites before they can cleave DNA but it has yet to be determined whether this is a general characteristic of the Type IIB endonucleases, or unique to BcgI. This question was examined here by analysing the reactions of several Type IIB restriction proteins on DNA molecules that have either one or two copies of the relevant recognition sequence. The enzymes were from commercial suppliers, at concentrations given in terms of units (U) of activity rather than molarity, and were initially studied under the conditions advised by the supplier (Table 2).
Results
BcgI
Previous studies had shown that the amount of BcgI protein needed to cleave a plasmid with two BcgI sites (pBR322) was smaller than that required to cleave a plasmid with one site (pUC19), so it was suggested that BcgI needs to interact with two copies of its recognition site to cut DNA.45 However, the activities of restriction enzymes can be affected by non-specific sequences flanking the specific site and, for enzymes with bipartite recognition sequences as is the case with BcgI (Table 1), by the sequence of the spacer within the site.17,51–53 While one of the two BcgI sites in pBR322 is the same as that in pUC19, the other differs in both flanking and spacer sequences. The enhanced activity of BcgI on pBR322 could thus be due to the context of the novel site. Furthermore, the different amounts of enzyme needed to cleave these two plasmids may reflect different affinities for each DNA rather than different reaction rates. A derivative of pUC19 was therefore constructed that had two BcgI sites, both in the same sequence context as the pUC19 site: pDG5 (Supplementary Data, Figure S1).18
When tested at a concentration of BcgI that resulted in the cleavage of almost all of both one-site and two-site plasmids, the initial rate for the reaction on pUC19 was found to be about sixfold lower than that on pDG5 (Table 3). The difference in reaction rates cannot be due to either sequence differences surrounding the specified bp of the recognition sequence, nor to insufficient enzyme for complete cleavage of either DNA. Instead, the data support the previous proposal45 that BcgI interacts with two copies of its recognition site, preferentially in cis, before cutting the DNA.
Proteins can mediate long-range communications between distant DNA sites by either tracking along the 1-D contour of the DNA from one site to the other without losing contact with the DNA; or by binding concurrently to both sites upon the juxtaposition of the sites in 3-D space, so looping out the intervening DNA.9,18 These routes can be distinguished by comparing a DNA catenane containing two interlinked circles with one site in each ring to a single circle of DNA with two target sites.33–35,54 The catenane cannot support 1-D tracking from one site to the other54 but, since the 3-D distance between two sites in the separate rings of a supercoiled catenane will be similar to that between two sites in a supercoiled plasmid,55 the catenane should be as effective as the two-site plasmid for 3-D looping.33 This strategy was applied previously to several other restriction enzymes with the plasmid pMLE2,34,35 a DNA with two target sites for numerous Type II enzymes, including BcgI, interspersed with two directly repeated res sites from the transposon Tn21. It can be converted by Tn21 resolvase into a catenane containing two interlinked rings of DNA with one BcgI site in each ring.
Initial rates for the reactions of BcgI on the parental plasmid with two BcgI sites (Figure 1(a)) and on the catenane with one BcgI site in each ring (Figure 1(b)) were measured from the decline in the concentration of the supercoiled substrates with time: the initial rates on the plasmid and on the catenane, 0.40 nM/min and 0.37 nM/min, respectively, were almost identical and in both cases considerably faster than that of 0.07 nM/min on a plasmid with one BcgI site under comparable conditions (data not shown). Moreover, almost all of the catenane was cleaved by BcgI in a highly concerted manner to give directly the final products with double-strand break(s) in both rings: neither the nicked forms, that would have arisen from single-strand breaks in one or both rings, nor the individual circles, from double-strand break(s) in the other ring, accumulated appreciably during the course of the reaction (Figure 1(b)). Hence, BcgI must bridge two copies of its recognition sequence through 3-D space and then introduce at least one double-strand break at each site before releasing the DNA.
AloI, BaeI and BplI
To examine whether other Type IIB endonucleases need to interact with two sites, a series of plasmids were constructed with either one or two copies of their recognition sequences (Supplementary Data, Figures S1–S3). Most of the constructs involved cloning a duplex of synthetic oligonucleotides at one location in the vector, to give a plasmid with one target site for each enzyme: then inserting into that plasmid at a separate location a second copy of the duplex, to give a plasmid with two copies of each target. In these cases, the recognition sites for the test enzyme on the one-site substrate, and both of its sites on the two-site substrate, were all within the same sequence context. On the two-site substrates made here, the distances between the pairs of sites varied from 673 bp to 1288 bp (Table 3; on pDG5, the BcgI sites are 1810 bp apart). As the reactions were carried out on supercoiled plasmids, this variation is not significant. In a supercoiled plasmid, the mean separation of sites in 3-D space is only marginally affected by their 1-D contour separation along the DNA.56 Furthermore, even the shortest separations span several persistence lengths, thus nullifying any effect due to twisting and bending of the intervening DNA.9 A further consideration for the test plasmid is that proteins capable of bridging two asymmetric DNA sequences are often affected by the orientation of the sequences: some prefer directly repeated sites, others sites in inverted orientation.40 This variation was removed by using as the new two-site substrates only plasmids with inverted sites.
Initial tests were carried out on three Type IIB enzymes, AloI, BaeI and BplI, as these embody several different aspects of the IIB systems (Table 1). Both BaeI and BplI contain two polypeptide chains, A and B, but these are present in BplI in a 1:1 rather than the 2:1 ratio seen with BcgI.49,50 Conversely, AloI is composed of a single polypeptide that carries endonuclease, methyltransferase and DNA recognition functions.47 BplI also differs from most Type IIB enzymes in having a palindromic recognition site while both BaeI and AloI have asymmetric targets, as is usual amongst IIB enzymes. Indeed, the recognition sequence for BaeI is the most asymmetric of the IIB sites; one segment of its bipartite sequence is 5 bp long, the other only 2 bp (Table 1). A plasmid with solitary recognition sites for AloI, BaeI and BplI, pJM1, was constructed and this used in turn to make a plasmid, pJM2, with two sites for each of these enzymes. Both AloI and BaeI cleaved the plasmid with two copies of their respective sites more rapidly than the plasmid with one copy (Figure 2(a) and (b)): fourfold faster in the case of AloI, similar to BcgI; by a larger factor, 13-fold, in the case of BaeI (Table 3). In contrast, BplI cleaved the supercoiled plasmids with one and two BplI sites at identical rates (Figure 2(c)).
Another strategy to determine whether a restriction enzyme needs two sites is to see if it can be activated to cleave a plasmid with one cognate site by an oligoduplex that has the recognition sequence.20,27 Interactions spanning separate molecules of supercoiled DNA are generally disfavoured so the interaction in trans needed to cleave the one-site plasmid can, at an appropriate concentration of duplex, be achieved more readily between plasmid and duplex than between two molecules of plasmid.24,25 However, higher concentrations of the duplex will inhibit plasmid cleavage as the enzyme becomes fully occupied with the duplex.24,39 When this test was applied to BaeI (Figure 3(a)), a specific oligoduplex with the cognate sequence for BaeI enhanced the cleavage of the plasmid with one BaeI site when present at relatively low concentrations, but abolished plasmid cleavage at higher concentrations. A control non-specific duplex lacking the sequence for BaeI failed to activate cleavage of the one-site plasmid, though high concentrations of this duplex led to a small reduction in plasmid cleavage, presumably due to the non-specific sequence acting as a weak inhibitor. Similar results were obtained when specific and non-specific duplexes were added to AloI reactions (data not shown). With BplI (Figure 3(b)), the non-specific duplex again had no effect on the cleavage of the one-site plasmid but in this case the specific duplex with the BplI site acted solely as an inhibitor of plasmid cleavage: no activation was observed at any duplex concentration tested.
Taken together, the above data show that both AloI and BaeI need to interact with two copies of their recognition sites in order to cut DNA. The faster reaction rates of these enzymes on the two-site plasmid, relative to the one-site DNA, are likely due to the intrinsic preference for interactions in cis, spanning sites in the same molecule of DNA, over interactions in trans, across separate molecules. Nevertheless the reactions of both AloI and BaeI on the one-site plasmid could be activated by adding a duplex with the requisite recognition sequence. These two enzymes are therefore not limited to sites in cis, but can also become active by binding two sites in trans. In contrast, the reactions of BplI on one and two-site plasmids, and the effect of a specific duplex on its one-site reaction, all match the expectations of a restriction enzyme that acts at individual sites, like EcoRV or BglI.17–19
Other Type IIB enzymes
The above experiments raise two possibilities. One is that the requirement of two recognition sites, as seen with BcgI, BaeI and AloI, is a general property of the Type IIB enzymes and that BplI is a solitary exception. The other is that the requirement for two sites is shared by only a fraction of the Type IIB enzymes and that BplI represents a major portion of these enzymes. Further plasmids with one or two copies of the requisite recognition sites were constructed to test most of the other Type IIB enzymes currently available (Table 1): AlfI, BsaXI, CspCI, FalI, PpiI and PsrI. When tested under the recommended reaction conditions for the enzyme in question (Table 2), four out of the six additional enzymes, AlfI, CspI, FalI and PpiI, cleaved their two-site substrate at significantly faster rates than their one-site substrate: by factors that varied from sixfold with AlfI and FalI to 43-fold with CspCI (Table 3).
AlfI and FalI recognise palindromic sequences, a feature of the BplI system but not of many other Type IIB systems (Table 1). Hence, Type IIB enzymes with palindromic sites do not necessarily act like BplI. The reactions of FalI on the plasmid with one FalI site were also examined in the presence of varied concentrations of oligoduplexes with and without its recognition sequence (data not shown). The specific oligoduplex activated FalI-cleavage of its one-site substrate, exactly like BaeI (Figure 3(a)), while the non-specific DNA had no effect apart from a slight inhibition at high concentrations, again like BaeI. FalI is therefore a Type IIB enzyme with a palindromic recognition sequence but which has to interact with two copies of its sequence to cut DNA, as is also AlfI (see below; Figure 5(a)).
Under their standard reaction conditions, the remaining two enzymes, BsaXI and PsrI, cleaved their two-site plasmids at rates that were ≤2-fold faster than those on their one-site substrates (Table 3; Figure 4(c)). A restriction enzyme that acts at individual sites will cleave a two-site DNA twice as fast as a one-site DNA if the DNA concentration is above that of the enzyme but below the Km value, simply due to the twofold difference in substrate concentration (i.e. recognition sites). Alternatively, if present at a >2-fold excess over the DNA, an enzyme that acts at individual sites will again carry out its initial reaction on a two-site DNA twice as fast as on a one-site DNA, as two molecules of the enzyme can then act simultaneously on the two-site DNA, one at each site. However, Type II restriction enzymes that need two sites often cleave their one and two-site substrates at similar rates in reactions at low ionic strength, but cleave the two-site DNA faster than the one-site DNA at high ionic strength.16,18,26,39 Consequently, to find out why BsaXI and PsrI cleave their one and two-site substrates at similar rates, and why BplI cleaves its equivalent substrates at identical rates, the reactions of these three enzymes were studied in buffers of varied ionic strength (Figure 4; Table 3).
The recommended reaction buffer for BplI contains 66 mM KOAc (Table 2) and, under these conditions, BplI cleaved to completion its one and its two-site substrates at identical rates (Figure 2(c)). An increase in the concentration of KOAc to 200 mM again allowed complete cleavage of both substrates, even more rapidly than at 66 mM, but a further increase to 800 mM led to incomplete cleavage of both substrates (Figure 4(a) and (b)). However, at all KOAc concentrations tested, the progress curves for the BplI reactions on its one and two-site substrates were superimposable. Similar results were obtained when the ionic strength was varied by adding NaCl instead of KOAc: the addition of NaCl affected both the rates and amplitudes of the BplI reactions, but NaCl had the same effect on the one-site and the two-site reactions (data not shown). In contrast, while PsrI had cleaved its two-site DNA at a marginally higher rate than its one-site DNA in a reaction buffer with 66 mM KOAc (Figure 4(c)), it cleaved the two-site DNA at a considerably faster rate than the one-site DNA in the presence of 200 mM KOAc (Figure 4(d)): 12-fold faster instead of the twofold difference under standard conditions (Table 3). For BsaXI, the ratio of its reaction rates on its two and one-site substrates likewise increased as the KOAc concentration was raised: from 1.6-fold at 50 mM to fourfold at 200 mM (Table 3).
Modes of action
Apart from BplI, all of the Type IIB enzymes tested here are capable of cleaving plasmids with two cognate sites more rapidly than plasmids with a single site, so they must act on the two-site DNA by bridging sites in cis. Among the restriction enzymes that bridge DNA sites in cis, the Type IIE systems first cleave one site and then in a separate reaction the other, often at a much slower rate.20,21 In contrast, the Type IIF enzymes act concertedly on two-site substrates and liberate as their initial product the DNA cut in both sites, bypassing intermediates cut at one site.19,22,24 These mechanisms can be distinguished by examining the transient intermediates formed during their reactions on a supercoiled (SC) DNA with two recognition sites.9,25,33 Such a DNA can be cleaved to give, successively: the open-circle (OC) form cut in one strand at one or both sites; the full-length linear (FLL) form cut in both strands at one site; and, as the end products, the two linear fragments after cutting both sites. If a restriction enzyme cleaves the DNA by means of separate but kinetically equal reactions at each site, the amount of the FLL form should rise to a maximum of 40% of the total DNA before declining, upon cleavage of the second site.17,57 If it cleaves one site more rapidly than the other, as is the case with Type IIE enzymes,30,33 the amount of the FLL form will reach a maximum of >40% of the total. Conversely, if the enzyme first cuts one site at a slow rate and then the second site at a faster rate, as is the case with the Type IIF enzymes,16,24 the maximal amount of FLL DNA will be <40% of the total. Three examples of this strategy are shown: AlfI (Figure 5(a)), CspCI (Figure 5(b)) and BcgI (Figure 1(a)).
In all three examples, and also with all of the other Type IIB enzymes examined here apart from AloI (data not shown), their reactions yielded none of the OC form of the DNA that would have arisen if products cut in one strand, at one or both sites, had accumulated during the reaction. A SC DNA with two recognition sites for a Type IIB enzyme has eight scissile phosphodiester bonds, in top and bottom strands on either side of both sites. Cleavage of any one of these eight bonds will convert the SC to the OC form but if the next phosphodiester bond to be cleaved is chosen at random from the seven remaining targets, six out of the seven leave the DNA in its OC state and only one, that opposite the initial nick, will result in the OC form being converted to the linear product with one double-strand break. Hence, Type IIB enzymes might be expected to generate large amounts of the OC intermediate before creating any linear product. The observation that no OC DNA accumulated shows instead that, after cutting one strand at a single locus, Type IIB enzymes must cut the second strand at that locus very shortly thereafter, within the lifetime of a single DNA–enzyme complex.
The reaction of the AlfI endonuclease on its two-site substrate yielded not only, as noted above, none of the OC form but also very little of the FLL form (Figure 5(a)). (No distinction is made here between the products from bilateral as opposed to unilateral cleavages at individual sites: see Materials and Methods.) The amount of FLL DNA reached a maximum at about 15% of the total, far below the value of 40% expected for kinetically equal reactions at each site. The DNA cut at one AlfI site is therefore almost always cleaved at the other site before being liberated. Hence, after bridging two copies of its recognition sequence, AlfI acts concurrently at both sites in a highly concerted manner. The reaction profile of AlfI is thus reminiscent of SfiI and the other Type IIF restriction enzymes.16,24,33 Several other Type IIB enzymes also converted major fractions of their two-site substrates directly to the final products with double-strand breaks at both sites, liberating none of the OC and only small amounts of the FLL intermediate: they included FalI, PpiI and PsrI (data not shown). AlfI and the other Type IIB enzymes that act in this manner therefore fall into the Type IIF category.
On the other hand, the CspCI endonuclease converted the SC substrate with two CspCI sites first to the FLL form and only later, after a lag phase, to the two linear products cut at both sites: as before, intermediates cut in one strand were not detected (Figure 5(b)). Hence, CspCI first cuts both DNA strands at one recognition site, and then in a separate reaction, creates one or more double-strand breaks at the other site. The amount of FLL DNA formed during the CspCI reaction reached a maximum of ∼40% of the total DNA. This level matches the expectation for a restriction enzyme that cleaves a two-site substrate in separate but equal reactions at each site.17,57 AloI, BaeI and BsaXI also cleaved their two-site substrates to give first the FLL linear form, again to a maximal yield of ∼40% of the total, and only later the linear fragments with double-strand breaks at both sites. Though these enzymes all need to bind two copies of their recognition sites to become fully active, they cleave each site in separate reactions.
In the reaction of BcgI on pMLE2, a plasmid with two BcgI sites (Figure 1(a)), the FLL form of the DNA was observed as a transient intermediate but even at its peak, the FLL species constituted <30% of the DNA, below the level indicative of two separate but equal reactions. This shows that BcgI sometimes dissociates from this plasmid after cutting one site but sometimes cuts both sites before dissociating. Other plasmids that have two BcgI sites, but with different flanking/spacer sequences from those on pMLE2, are cleaved without releasing any of the FLL DNA (S. Ganguly, J.J.T.M. & D.M.G., unpublished results). Moreover, BcgI cleaved the catenane derived from pMLE2 in a highly concerted manner: it released virtually none of the individual circles that would have been liberated by making a double-strand break in one ring (Figure 1(b)). Hence, BcgI rarely dissociates from the catenane after cutting just one site and instead remains bound until it has opened both rings. Other restriction enzymes were observed previously to act more concertedly on catenanes with one target site in each ring than on plasmids with two sites in cis.33 A synaptic complex between two sites in the separate rings of a catenane is likely to be more stable than one spanning sites in cis, as the juxtaposition of the sites in the catenane will not impose any deformation of the intervening DNA. The enzyme may therefore be able to cut both sites within the lifetime of the catenane synapse but not within the lifetime of the plasmid synapse.
Amongst the Type IIB enzymes that cleave plasmids with two cognate sites more rapidly than plasmids with one target, the largest differences observed under standard reaction conditions were with BaeI and CspCI, which cleaved their two-site substrates 13 and 43-fold faster, respectively, than their one-site substrates (Table 3). Yet these two enzymes cleaved two-site plasmids by means of separate but kinetically equal reactions at each site (Figure 5(a)). To determine how these two enzymes communicate between distant DNA sites, their reactions were studied on catenane substrates that had in each ring either one BaeI site or one CspCI site (data not shown). When tested with the relevant enzyme, the catenanes were cleaved at similar rates to the parental two-site plasmid for that enzyme, in both cases more rapidly than the corresponding one-site plasmid. Moreover, like BcgI (Figure 1(b)), both BaeI and CspCI cleaved the catenanes much more concertedly than the plasmids: they released almost none of the individual circles from cutting the catenane in a single ring. Hence, both BaeI and CspCI act rapidly on DNA with two copies of their recognition sites by first spanning the sites through 3-D space, as opposed to tracking along the DNA. They can then act concertedly at both sites. BaeI and CspCI thus also belong in the Type IIF category of restriction endonucleases.
Discussion
The general case
The Type IIB endonucleases constitute a discrete subset of the Type II R/M systems,2 delineated by their bipartite recognition sequences and by their bilateral sites of DNA cleavage either side of their recognition sites (Table 1). To determine whether Type IIB enzymes need to interact with two target sites in order to cleave DNA, the reactions of ten IIB nucleases were examined on plasmids that carry either one or two copies of the requisite sequence. With one exception, BplI, they all cleaved their two-site substrates more rapidly than the one-site substrates (Table 3) though two, BsaXI and PpiI, gave only twofold (or smaller) differences under their standard reaction conditions. These two both exhibited larger differences at elevated ionic strengths (Figure 4, Table 3). Moreover, all of the Type IIB enzymes tested against catenanes cleaved the catenane with one site in each ring as readily as the parental plasmid with two sites in a single ring (Figure 1). In addition, in all cases tested apart from BplI, the Type IIB enzymes cleaved plasmids with a single cognate site at enhanced rates in the presence of cognate oligoduplexes (Figure 3).
These experiments confirm the original proposal of Kong & Smith,45 that BcgI interacts with two copies of its recognition sequence before cutting DNA, and show further that the vast majority of the Type IIB enzymes act in the same way. Proteins that interact with distant DNA sites intrinsically prefer sites in cis over sites in trans,9,24,25 which accounts for why these enzymes cleave the two-site plasmids more rapidly than their one-site substrates. Nevertheless, the catenane experiments show that they capture their two sites in cis through 3-D space and not by tracking along the DNA, like the Type I enzymes.34,54 They can, however, also act in trans, by forming a complex bridging one recognition site on a plasmid and another on an oligoduplex.
The Type IIB endonucleases that cleave DNA after binding two cognate sites include some composed of two polypeptide chains, such as BcgI and BaeI,42,49 and some composed of a single polypeptide, such as AloI and PpiI.46–48 They also include some with asymmetric recognition sequences, like BsaXI, CspCI and PsrI, and some with palindromic sites, like AlfI and FalI. Furthermore, they include some that require AdoMet for their nuclease activities, viz. BcgI and CspCI, and others with no requirement for AdoMet, viz. BsaXI and PsrI (Table 1). The need to interact with two sites before cutting the DNA is thus a general property of the Type IIB enzymes, common to a wide range of these enzymes even though they differ from each other in many other respects. Two main mechanisms exist for the Type II restriction enzymes that use two sites:9,19–21,33 some, the Type IIE systems, employ one site as an activator to enhance cleavage of another site;27–30 others, the Type IIF systems, act concertedly at both sites.22,24,32–35 The Type IIB nucleases fall into the latter category.
This study extends the range of restriction endonucleases that are currently known to cleave DNA only after interacting with two target sites. In addition to the Type I, III and IV systems,1,4,5 which constitute ∼60% of the R-M systems found in nature,3 the set includes a substantial fraction of the Type II enzymes: a portion of those that act within palindromic sites;18 the majority of the Type IIS systems that act on one side of an asymmetric site;36–40 and now, from this study, essentially all of the Type IIB enzymes that act bilaterally either side of a bipartite sequence. The enzymes that need two sites may therefore constitute 80% of the total and those that act at individual sites only 20% of the restriction enzymes present in vivo, even though the latter include the so-called archetypes like EcoRI, EcoRV and BamHI, the enzymes most widely used as tools for DNA manipulations in vitro.58,59 Though enzymes acting at individual sites might seem to be more efficient at restricting DNA than those requiring two sites, the widespread requirement for two sites indicates an advantage over the one-site systems.19 The need to interact with two copies of the recognition sequence before cleaving the DNA, rather than a single copy, may function as a double-check to ensure these enzymes only cleave DNA in response to the cognate sequence, and thus avoid untoward reactions at non-cognate sites.9,38,60 However, other restriction enzymes can utilise a cognate site to initiate cleavage of a non-cognate site.22,35,61,62 In these cases, the bridging interaction between cognate and non-cognate sites serves to broaden the range of sequences that the enzyme cleaves.61
The special case
On the other hand, one of the ten Type IIB enzymes tested, BplI, showed none of the characteristics of a restriction enzyme that interacts with two sites. Instead, BplI catalyses separate and independent reactions at each copy of its recognition site, in the same manner as the standard restriction enzymes like EcoRV.19 Under all reaction conditions employed with BplI, the progress curves for the utilisation of its one-site and two-site substrates were superimposable, even though its overall activity varied considerably over the range of conditions tested (Figures 2(c), 4(a) and (b)). Unlike Psr I for example, which cleaved its one and two-site substrates at similar rates at low ionic strength but at dissimilar rates at higher strengths (Figure 4(c) and (d)), no difference was observed with BplI even when the salt concentration was too high to allow complete DNA cleavage. Moreover, BplI cleaved the plasmid with two BplI sites by means of separate reactions at each site, albeit at the same rate (data not shown). In addition, an oligoduplex with the recognition sequence for BplI inhibited rather than enhanced its cleavage of a plasmid with one BplI site (Figure 3(b)), which again indicates that BplI can bind only one copy of its recognition sequence at a time.
BplI clearly acts in an atypical manner for a Type IIB endonuclease, but its unusual properties cannot be due to its palindromic recognition sequence. Though most Type IIB enzymes recognise asymmetric sites (Table 1), some such as AlfI and FalI have palindromic sites yet still need two sites to cleave DNA. However, the unique properties of BplI might be a consequence of its subunit composition. Like BcgI, BplI is made up of two polypeptide chains but seemingly in a 1:1 ratio instead of the 2:1 ratio reported for BcgI.42,50 A related possibility is that BplI is not really a Type IIB enzyme, and is instead a novel Type IIS system with a palindromic recognition site. The restriction enzymes currently considered as Type IIS systems all have asymmetric sites and cut DNA at specified positions on one side of the site.2,3 A protein must bind to an asymmetric sequence in one particular orientation, which fixes the position of its catalytic residues on the DNA,57 but it can bind to a palindromic site in either of two orientations. BplI cleaves DNA either side of its site 8 and 13 bases away (Table 1)50 so on 50% of the occasions that it binds to its site, its catalytic functions may be located 8/13 bases to the “left” of the site and, on the 50% of the time, 13/8 bases to the “right” of the site. A Type IIS enzyme with a palindromic site would thus make bilateral cleavages either side of its site in the characteristic manner of a Type IIB enzyme. Though most of the Type IIS enzymes need to interact with two sites for full activity,13,37–40 some need only one site.36
Materials and Methods
Enzymes
Tn21 resolvase was purified as before.33,34,54 All other enzymes were obtained from commercial suppliers, stored at –20 °C and used essentially as advised by the supplier. The suppliers of the Type IIB restriction enzymes studied here, and the primary reaction buffers for each enzyme, are listed in Table 2. The concentrations of these enzymes were given in terms of units (U) of enzyme activity per ml, as specified by the supplier. While all of the commercial enzymes were free from non-specific nucleases, the purities of the preparations were not known and could not be assessed here. Most of the supplied samples contained insufficient amounts of protein for further analysis: the number of units in each purchase varied from 50 U to 1250 U and Type II restriction enzymes typically have specific activities of 106–107 U/mg of protein.10,19
DNA
The plasmid pUC1963 and its derivatives pMDS2(a),34 pMLE1 and pMLE2,33 and pDG5,18 have been described. Specifically, pDG5 had been constructed from pMLE1 by inserting a 56 bp duplex that contained the same sequence as that around the BcgI site in pUC19: pDG5 is thus a 3806 bp plasmid with two BcgI sites 1810 bp apart that are identical to each other and to that in pUC19.
Further plasmids, with one or two copies of Type IIB recognition sites were constructed from pDG5 and pMDS2a (Supplementary Data, Figures S1–S3). Typically, the vector was first linearised with a restriction enzyme and then ligated to a DNA duplex with appropriate single-strand extensions: the duplexes carried recognition sequences for several Type IIB enzymes and were made by annealing pairs of synthetic oligodeoxyribonucleotides (MWG Biotech). The ligation mixtures were used to transform Escherichia coli HB101 and the plasmids from several transformants sequenced across the site of the insertion (University of Dundee Sequencing Service). From each ligation, some transformants were found to contain a plasmid with a single copy of the insert at the requisite site. These plasmids were then used as vectors to clone a second duplex at a separate site, to give plasmids with two inserts in either inverted or directly repeated orientation. The constructs with inverted (head-to-head) inserts were used as the two-site substrates.
The transformants were cultured in M9 minimal media containing 37 MBq/l [methyl-3H]thymidine (GE Healthcare) and the plasmids purified by CsCl density-gradient centrifugations.31–34 The preparations generally contained 80–95% supercoiled monomeric plasmid, with 5–20% open-circle and dimeric forms. The supercoiled forms of pMLE2, pJM2 and pIS2 were converted into catenanes by reactions with Tn21 resolvase at a 24-fold molar excess over the plasmid, as noted before.18,33,54 DNA concentrations were evaluated from A260 measurements.
Reactions
Reactions were usually carried out by adding 0.5–4 μl of the restriction enzyme (diluted, if necessary, in the buffer advised by the supplier) to 200 μl of 3H-labelled DNA (5 nM) in the appropriate buffer at the requisite temperature (Table 2). One 10 μl aliquot (the zero-time point control) was removed before adding the enzyme and further aliquots at timed intervals thereafter. The aliquots were mixed immediately with 5 μl of an EDTA Stop-Mix.24,32,38 In some instances, reactions were carried out in 20 μl volumes containing constant amounts of enzyme and plasmid but with varied concentrations of an oligoduplex: some duplexes contained, others lacked, the recognition sequence for the test enzyme. After a fixed time interval, these reactions were quenched with Stop-Mix as described above.
The quenched samples were analysed by electrophoresis through agarose.26,40 From reactions on plasmid substrates, the following forms of the DNA were separated: the intact supercoiled (SC) plasmid; the nicked open-circle (OC) form; the full-length linear (FLL) DNA cut in both strands at one site; and, for substrates with two sites, the two linear products (L1 and L2) from making double-strand breaks at both sites.16,25 With catenane substrates, the intact catenane was separated from the catenanes with nicks in one or both rings; and from both the separate rings and the linear products due to double-strand breaks in one or both sites.7,33–35 Type IIB enzymes cleave DNA on both sides of their recognition sites, but the linear product from cutting on one side of a site is only 27– 33 bp longer than that cleaved on both sides of the same site. As the plasmids used here were ∼4 kb in size, with ∼1 kb between the sites in the two-site substrates, the products from bilateral cleavages were not separated from those with unilateral cleavages. Moreover, the 27–33 bp fragments excised from the remainder of the DNA were too small to detect on the agarose gels used here. It was assumed that DNA products cut in both strands at one or both recognition sites had been cleaved bilaterally at those sites.
Segments of the gels were analysed by scintillation counting,25,40,57 to determine the concentration of each form at each time point. The concentrations shown in Figures 1–5 are the means from three independent experiments: for clarity, the error bars for the standard deviations (typically <10% of the mean values) have been omitted. Initial rates were evaluated from the mean values for the concentration of supercoiled DNA, by using GRAFIT (Erithacus Software, Horley, UK) to fit to a linear slope the decrease in these concentrations with time, starting at time zero and extending over the zero-order stage of the reaction: the errors in the reaction velocities cited in Table 3 are the standard errors from the fitting procedure.17,57 The errors on the ratios of the rates on the one and two-site plasmids were calculated from the error limits on each of the individual rates.
For the majority of the Type IIB enzymes tested here, the concentrations of their substrate ([S]) declined linearly during their reactions, for ≥50% of the total reaction: for example, BaeI (Figure 2(b)). However, for some such as AloI (Figure 2(a)), [S] declined in an exponential rather than a linear fashion. For substrate utilization to follow an exponential curve, one of the following must pertain: the reaction contains [E0] > [S] and thus has the characteristic first-order kinetics for a single-turnover reaction; or it is under multiple-turnover (steady-state) conditions with [E0] < [S], but with an initial [S] below the Km value so that the reaction velocity then declines in parallel with [S].52 The former may apply to BcgI, as this enzyme seems to act stoichiometrically rather than catalytically.42,45 Further experiments using BcgI that had been purified in this laboratory (S. Ganguly, S. Milsom, J.J.T.M & S.E.H., unpublished results) show that all of the BcgI reactions described here contained enzyme in excess of the substrate. It has yet to be determined which of these two possibilities apply to the other Type IIB enzymes that give exponential curves, as their concentrations were known only in terms of enzyme units, not molarities. On the other hand, a linear decline in [S] can only arise from a zero-order steady-state reaction involving multiple turnovers of the enzyme. Consequently, to permit comparisons of different enzymes, all of the reaction rates were evaluated as zero-order velocities from the decline in [S] with time over the initial portion of the reaction.

koac, mg(oac)2 and tris-oac, potassium, magnesium and tris acetate, respectively