However, some enzymes that act at palindromic sites are monomers and other tetramers. EcoRI, EcoRV, NotI, and BglI ( Table 1) all act in this way. These enzymes bind to their recognition sites in symmetrical fashion, with each subunit making the same contacts to the DNA and with the active sites in the two subunits, each positioned to cleave its target phosphodiester bond in one DNA strand. The restriction endonucleases that recognize palindromic DNA sequences, the type IIP enzymes, are usually dimers of identical subunits. Halford, in Brenner's Encyclopedia of Genetics (Second Edition), 2013 Mechanisms of DNA Recognition and Cleavage Similar folds have since been found in many other proteins that act on DNA, including enzymes involved in replication, recombination, repair, and transposition.
In some cases such as EcoRV, recognition of the specific sequence also depend on deformations to DNA structure.ĭespite the overall dissimilarity in their amino acid sequences and their differences in their structural elements for DNA recognition, some Type II restriction enzymes have similar tertiary structures in terms of the overall fold of the polypeptide chains, for example, EcoRI has a similar fold to BamHI and likewise EcoRV to PvuII. These also differ considerably from one enzyme to the next. Instead, their DNA-recognition elements encompass a wide variety of nonstandard structures, including loops, sheets, and helices. However, the restriction enzymes do not usually employ the elements of protein structure that are common among DNA-binding proteins, such as the helix–turn–helix or the zinc finger. As with most other proteins that act at specific DNA sequences, restriction enzymes recognize their target sequences primarily by multiple hydrogen bonds to the bases in the major groove of the DNA. The structures of numerous Type II endonucleases, and their complexes with DNA, have been determined by X-ray crystallography. One active site in the dimer is positioned against the scissile phosphodiester bond in one strand of the DNA and likewise the second active site on the other strand. They interact with their recognition sequences symmetrically so that all the contacts between one subunit of the protein and one-half of the recognition site are duplicated by the second subunit with the other half of the DNA. The enzymes that recognize palindromic DNA sequences are generally dimers of identical subunits. Halford, in Encyclopedia of Biological Chemistry (Second Edition), 2013 Protein Structures
It is important to note, however, that the cruciform formation of palindromic DNAs in vivo remains to be fully validated. Replication origins are often associated with longer palindromes of several dozen base pairs, and it has been proposed that cruciform-binding proteins, such as members of the 14-3-3 protein family in mammals, bind to these structures at palindromic sequences at or near potential replication origins and are involved in the initiation of DNA replication. However, there is no evidence that such short palindromes adopt a cruciform conformation. Short palindromic DNA sites are often recognized by specific protein dimers such as restriction enzymes, methylases, or other DNA-binding proteins. There are many existing hypotheses for the biological roles of the palindromic DNAs that potentially extrude cruciform DNA structures. There is also a report of cruciform formation following the artificial introduction of negative superhelical tension into a specific DNA region. However, several additional studies have now provided some evidence of cruciform formation in living cells using chemical fixation methods. Some early experiments have suggested that DNA superhelices in living cells may not be sufficient to extrude cruciform structures and that the kinetics of cruciform formation under physiological conditions is generally considered to be too slow to fulfill a biological role in living cells. Using the detection methods described above, a number of attempts have been made to detect cruciform conformations in living cells. Whether cruciform extrusion of palindromic DNA occurs in vivo remains controversial. Kurahashi, in Brenner's Encyclopedia of Genetics (Second Edition), 2013 Biological Roles of Cruciform DNA In Vivo