Restriction Endonucleases Flashcards
➜ degrade DNA molecules by breaking the
phosphodiester bonds that link one nucleotide to the
next in a DNA strand
➜ may be specific for DNA or RNA, such as DNases or
RNases
Nucleases
Kinds of Nucleases
- Endonucleases
- Exonucleases
hydrolyze
internal bonds within a
polynucleotide chain
Endonucleases
remove
nucleotides one at a time from
the end of a DNA molecule
Exonucleases
➜ made in early 1950s
➜ some strains of bacteria are immune to bacteriophage
infection, a host defense mechanism.
➜ bacterium produces an enzyme that degrades the
phage DNA before it has time to replicate and direct
synthesis of new phage particles
Restriction Endonucleases
➜ sequences recognized by REs read the same from left
to right as they do from right to left on the
complementary strand.
➜ Palindromic sequence
Palindrome
➜ exhibit both restriction and DNA modification activities
➜ require the cofactors such as Mg2+ ions, Sadenosylmethionine (SAM) and ATP for their activity
➜ the recognition sequences are quite long with no
recognizable features such as symmetry
(asymmetrical).
➜ cleave DNA at nonspecific sites and that can be 1000
base pair or more from recognition sequence
Type I Restriction Enzymes
➜ because the methylation reaction is performed by the
same enzyme which mediates cleavage, the target
DNA may be modified before it is cut.
➜ Because of these features, the type I systems are of
little value for gene manipulation.
Type I Restriction Enzymes
_____ and their corresponding modification
methyltransferases act as separate proteins
➜ recognition site is 4-6 bp sequence, usually
palindromic
Type II Restriction Enzymes
restriction and modification are mediated by separate
enzymes so it is possible to cleave DNA in the absence of
modification.
➜ Advantages over type I and III systems
- the restriction activities do not require cofactors such as
ATP or S-adenosylmethionine, making them easier to
use. - require only Mg2+ ions as cofactors
- are site-specific as they hydrolyze specific
phosphodiester bonds in both DNA strands. - Generally used as the key material in molecular biology
and recombinant DNA techniques including genome
mapping, RFLP analysis, DNA sequencing and cloning
Type II Restriction Enzymes
staggered ends on a DNA molecule with short, singlestranded overhangs.
- Sticky 5’ ends (5’ overhang)
- Sticky 3’ ends (3’ overhang)
Sticky ends
have straight cut, down through the DNA, that results
in a flat pair of bases on the ends of the DNA
Blunt ends
➜ possess both restriction and modification activities (same with
Type I).
➜ recognizes specific sequences and cleave 25-27 bp outside of
the recognition sequence, in a 3’ direction (asymmetrical)
➜ requires 2 restriction sites in opposite orientation
➜ requires Mg2+ ions for their activity
Type III Restriction Enzymes
➜ cleave outside of their recognition sequences.
➜ require two such sequences in opposite orientations
within the same DNA molecule.
➜ rarely give complete digests.
➜ recognize modified, typically methylated DNA.
Type IV Restriction Enzymes
