113
for generating single-stranded 3′-ends, on which terminal deoxynucleotidyl transferase (TdT) can act (Little 1981).
4.8.2 Restriction Endonucleases
Restriction endonucleases are enzymes that hydrolyze the phosphodiester backbone
of dsDNA at or near specific nucleotide sequences called restriction sites. Few
restriction enzymes have been found to be active on ssDNA, but with only low
efficiency. Majority of the restriction enzymes have been isolated from bacteria and
archaea, where they provide defense against bacteriophages. Phage DNA is digested
by restriction enzymes present in a prokaryote, but its own genomic DNA is protected by methylation of target sequence by DNA methyltransferase.
Restriction sites are usually between 4 and 8 bases long and many of them are
inverted repeat palindrome, where the base sequence on one strand when read forward is the same as the sequence on the complementary strand when read backward. Restriction enzymes make two cuts in the sugar-phosphate backbone of
dsDNA, one in each strand (Pray 2008). They are categorized into four types based
on their differences in structure, cofactors, restriction sequence specificity, and position of cleavage from the restriction site.
Type I restriction enzymes cleave DNA nonspecifically at positions far from their
recognition sequences and hence do not have much practical application. They
have both restriction and methylase activities and require ATP and
S-adenosyl-L-methionine.
Type II restriction enzymes form homodimers that cleave DNA within its restriction
site or at specific positions near it. They lack methylase activity. Most enzymes
in this class are ATP independent, but require Mg
2+
as a cofactor. Majority of the
commercially available restriction enzymes belong to this category and are routinely used for DNA analysis and molecular cloning. Of the 3500 different Type
II restriction enzymes that have been discovered, more than 600 are commercially available.
Type III restriction enzymes are hetero-oligomeric proteins that cleaves DNA at
about 20–30 base pairs away from the recognition site. They require two different nonpalindromic recognition sequences that are reversely oriented within the
same DNA molecule to carry out cleavage. AdoMet and ATP are essential for
DNA methylation and restriction digestion activities respectively.
Type IV restriction enzymes act preferentially on DNA molecules with modifications like methylation, hydroxymethylation, and glucosyl-hydroxymethylation
(Williams 2003).
4.8.2.1 Nomenclature
Restriction enzymes are usually named after their source of origin. The first three
letters of the name are abbreviations of the genus and species names of the organism. The fourth letter comes from the bacterial strain designation, and the Roman
4 Enzymes as Molecular Tools
for generating single-stranded 3′-ends, on which terminal deoxynucleotidyl transferase (TdT) can act (Little 1981).
4.8.2 Restriction Endonucleases
Restriction endonucleases are enzymes that hydrolyze the phosphodiester backbone
of dsDNA at or near specific nucleotide sequences called restriction sites. Few
restriction enzymes have been found to be active on ssDNA, but with only low
efficiency. Majority of the restriction enzymes have been isolated from bacteria and
archaea, where they provide defense against bacteriophages. Phage DNA is digested
by restriction enzymes present in a prokaryote, but its own genomic DNA is protected by methylation of target sequence by DNA methyltransferase.
Restriction sites are usually between 4 and 8 bases long and many of them are
inverted repeat palindrome, where the base sequence on one strand when read forward is the same as the sequence on the complementary strand when read backward. Restriction enzymes make two cuts in the sugar-phosphate backbone of
dsDNA, one in each strand (Pray 2008). They are categorized into four types based
on their differences in structure, cofactors, restriction sequence specificity, and position of cleavage from the restriction site.
Type I restriction enzymes cleave DNA nonspecifically at positions far from their
recognition sequences and hence do not have much practical application. They
have both restriction and methylase activities and require ATP and
S-adenosyl-L-methionine.
Type II restriction enzymes form homodimers that cleave DNA within its restriction
site or at specific positions near it. They lack methylase activity. Most enzymes
in this class are ATP independent, but require Mg
2+
as a cofactor. Majority of the
commercially available restriction enzymes belong to this category and are routinely used for DNA analysis and molecular cloning. Of the 3500 different Type
II restriction enzymes that have been discovered, more than 600 are commercially available.
Type III restriction enzymes are hetero-oligomeric proteins that cleaves DNA at
about 20–30 base pairs away from the recognition site. They require two different nonpalindromic recognition sequences that are reversely oriented within the
same DNA molecule to carry out cleavage. AdoMet and ATP are essential for
DNA methylation and restriction digestion activities respectively.
Type IV restriction enzymes act preferentially on DNA molecules with modifications like methylation, hydroxymethylation, and glucosyl-hydroxymethylation
(Williams 2003).
4.8.2.1 Nomenclature
Restriction enzymes are usually named after their source of origin. The first three
letters of the name are abbreviations of the genus and species names of the organism. The fourth letter comes from the bacterial strain designation, and the Roman
4 Enzymes as Molecular Tools