Restriction Endonucleases Produce Sticky Ends

Many Restriction Endonucleases Produce Sticky Ends  

Arrows indicate the phosphodiester bonds cleaved by the restriction endonucleases. The name of the restriction endonuclease consists of a three-letter abbreviation of the bacterial species from which it is derived—for example Eco for Escherichia coli. 

To make life more challenging for molecular biologists, some enzymes can also cut with less than absolute specihcity. This is called star ( ) activity and can often be seen if the enzyme concentration is too high or the enzyme is incubated with the DNA too long. 

As we will see in the next section, the production of sticky ends by a restriction enzyme is very important to the process of creating recombinant DNA. Once DNA is cut with a particular enzyme, the sticky ends ht back together. At that point it does not matter whether the pieces came from the original piece of DNA. In other words, pieces of DNA from different sources can be put together as long as they were both cut with the same restriction enzyme. 

Restriction endonucleases are enzymes produced by bacteria that hydrolyze the phosphodiester backbone of DNA at specific sequences. 

---

Fig. 6.18. Actions of restriction endonucleases EcoRI, Psfl, and Sma at their recognition sequences. Note that EcoRI and Pst enzymes produce "sticky ends, with overlapping sequences, whereas Smal produces blunt ends, or nonoverlapping sequences.

The cloning and manipulation of genes usually depends upon the precise cutting of DNA into discrete fragments by restriction endonucleases. Many restriction enzymes generate cohesive ends (sticky ends). Thus, EcoRI produces DNA fragments with the single-stranded "tails" shown here at the 5 -ends of the cut duplexes ... 

The MCS is a stretch of unique restriction endonuclease cleavage sites between the promoter and the poly (A) signal in expression plasmid vectors, and is used as a common area to insert a cDNA or gene of interest. By using the MCS, the genes of interest can be inserted and assayed with ease. Sticky-end and blunt-end vector strands are available at different restriction sites. Although the MCS is very convenient, it should be noted that these sites usually remain in the mRNA transcribed from the expression unit of the vector and may reduce its stability or the efficiency of translation. Furthermore, one must be careful not to produce an ATG codon at the junctional sequence between the MCS and the insert, because it may work as a false start codon in the resulting mRNA and interfere with the production of the correct product. 

Restriction enzymes (endonucleases) typically cut DNA at specific 4- to 8-bp palindromic sequences, producing defined fragments that often have self-complementary single-stranded tails (sticky ends). 

Why are sticky ends important Restriction endonucleases play a large role in the manipulation of DNA. These enzymes produce short, single-stranded stretches, called sticky ends, at the ends of cleaved DNA. The sticky ends provide a way to link DNAs from different sources, even to the point of inserting eukaryotic DNA into bacterial genomes. 

EXAMPLE 8.17 Among the most important enzymes of recombinant DNA technology are the restriction enzymes. These are endonucleases that cleave DNA only at specific sequences of bases (called restriction sites). Typically, restriction sites are palindromic, in other words, the sequences are the same in the 5 — 3 and 3 — 5 strands. Restriction enzymes are produced by bacteria as an antiviral defense, and they cleave the DNA of viruses (bacteriophages) that infect them. However, they do not cleave host bacterial DNA. Fig. 8-17 shows the restriction sites of three common restriction enzymes, BamHI, EcoRI, and PvuII. Because BamHI and EcoRI cleave their restriction site asymmetrically, they produce overhangs in the cleaved DNA, called sticky ends. Conversely, PvuII cleaves symmetrically, producing blunt ends. 

---