Restriction Enzmes

Restriction enzymes are very ordinary reagents used in the analysis and manipulation of DNA and are used in a variety of "gene technology" investigate programs and protocols. Most students now gather the aid of these enzymes lacking always learning or realizing their origins or their in vivo role. These clarification are intended to sort out solely with the intention of. Hopefully, if you understand their natural role and occurrence, you will be able to understand their attention more thoroughly.

There are several ways by which DNA is transferred from lone bacterial cell to a further (i.E., from a donor to a recipient).

1. Transduction - Phage are able to conveying viral and bacterial DNA in a species/strain point style.

2. Conjugation linking two bacteria.

3. Uptake of emancipated genetic material using crust proteins with the intention of get on to up competence systems and others.

Regardless of how DNA enters a recipient cell, once inside the cell, foreign DNA may either carry on (i.E., imitate and/or recombine with the recipient DNA) or it could be degraded. Degradation of the foreign DNA is termed restriction and is accomplished by nucleolytic cleavage. If the DNA survives, it is due to the modification endeavor of the donor cell.

Restriction Modification (RM) systems be inflicted with been found in bacteria. Whether they occur in other microbes or in eucaryotic cells is still uncertain. RM systems be inflicted with been found in Anabena, a blue-green algae (called Cyanobacteria by some). Not all bacteria be inflicted with RM systems and hence in persons with the intention of lack such systems, all genetic material expected survives.

RM systems are ordinarily named according to the bacteria in which it was found. These names are furthermore often used as their gene designations. Some ordinary examples of RM systems include:

NAME	SOURCE	RECOGNITION SITE
EcoR	E. coli	5' - G|AATTC - 3'
Ana II	Anabena	5' - G|G(A/T)CC - 3'
Hind III	Haemophilus	5' - A|AGCTT - 3'
Stu I	Streptomyces	5' - AGG|CCT - 3'
Sau3A1	Staphlococcus	5' - N|GATC - 3'

The vertical line in the recognition site sequence is the cleavage site. N= any base

The RM systems are species/strain specific thus only allowing the survival of DNA received from a like species or strain having the same RM system.

Any native double stranded DNA can serve as a substrate for the enzymes of RM systems provided that there are specificity sequences. DNA that is modified on only one strand of the duplex DNA is still preserved from restriction. Thus, semi-conservative replication of a cells DNA will not lead to self-restricion. Soon after replication, the new strand is quickly modified prior to the next round of replication.

THE ENZYMES:

Restriction is accomplished by sequence specific (site specific) endonucleases. These enzymes are able to cleave the interior of DNA strands (as opposed to exonucleases which cleave from a terminus). Once terminuses are produced by the internal cleavages of the endonucleases, a large variety of exonucleases can degrade the DNA very rapidly.

Modification is accomplished by sequence specific methylase enzymes. In any cell with a RM system, both the restriction and modification enzymes have the same sequence specificity.

Generally, the methylase activity is "replication independent" and the most common site for methlyation is the extra cyclic amine group of adnine. The methyl donor for the methylases is S-adenosyl methionine (SAM). Mg++ and ATP are not needed for the activity. The methylases can compete with the restriction enzymes (since their specificity sites are the same) and thus some portions of foreign DNA may be preserved. This is termed "leakiness" of the system or "rescue". In E.Coli, up to 3% of the foreign DNA may be salvaged in this manner.

THERE ARE AT LEAST THREE TYPES OF RM SYSTEMS (I,II & III)

TYPE I RM was the first type discovered but is not the simplest nor the most common. In this type, the restriction modification is accomplished by a five peptide complex - R2M2S or 2:2:1 (R:M:S)

HsdR -----> R (135kd), hsdM -----> M (62kd), hsdS -----> S (55kd)

Mutants of gene hsdR are designated r-m+, etc.

Examples are EcoK and EcoB.

Type I systems recognize a bipartite asymetrical sequence:

---TGA*NnTGCT--- * = methylation sites

---ACTNnA*CGA--- n = 6 for EcoK, n = 8 for EcoB

The S peptide is the site recognition factor and may control protein to protein specific assembly of the complex. SAM must be bound to the M protein for S to recognize the site.

The cleavage site is about 1000 base pairs from the recognition site where the RMS complex is bound. This is accomplished by a "looping" mechanism and cleavage requires ATP. The strands are cleaved one at a time.

TYPE II RM systems are the simplest and most common (at least commercially). In this system, there are separate R and M proteins each able to recognize the cleavage site (no S protein needed). Each may compete directly and no complex is formed. Site recognition is via short palindromic base sequences that are 4-6 base pairs long. Cleavage is at the recognition site (but may occasionally be just adjacent to the palindromic sequence, usually within) and may produce blunt end terminuses or staggered, "sticky end" terminuses. No ATP requirements have been detected for the endonuclease activity.

TYPE III RM systems are interesting in that the M and S function is found in one protein labeled MS (hsdMS => 73-80kd in E.Coli) while the restriction is accomplished by the hsdR gene product R (hsdR => 106-110kd in Ec). The recognition site is a 5-7 bp asymmetrical sequence. Cleavage is ATP dependent 24-26 base pairs downstream from the recognition site and usually yields staggered cuts 2-4 bases apart.

TYPE I	TYPE II	TYPE III
Separate endonuclease & methylase proteins	Bifunctional enzyme of 3 subunits, R,M & S (2:2:1)	Bifunctional enzyme of 2 subunits, MS & R
Short palindrome 4-6 bp recog. Site, same for R & M	Recog. Site = bipartite, asymetrical sequence = 5-7bp	Recog. Site = asymetrical  sequence = 5-7bp.
Cleaves at recognition site. Yields blunt or sticky end, within or next to site.	Cleaves 1000 bp from recognition site	Cleaves 24-26bp downstream of recognition site, staggered cut 2-4 bases apart.
R & M act separate	R & M may compete or may be exclusionary.	R & M occur simultaneously & may compete.
No ATP needed for R	Needs ATP for R	Needs ATP for R