3' GT Py Pu A C 5' 5' CA Pu Py T G 3'
is the restriction site of which enzyme ?

Question

Accepted Answer

The correct option is 3: Hind II

The enzyme Hind II recognizes the sequence $	ext{3'} 	ext{ G T P y P u A C } 	ext{5'}$ on one strand, which corresponds to $	ext{5'} 	ext{ C A P u P y T G } 	ext{3'}$ on the complementary strand, where 'Py' stands for any Pyrimidine base (Cytosine or Thymine) and 'Pu' stands for any Purine base (Adenine or Guanine). Specifically, the recognition sequence for Hind II is $	ext{5'} 	ext{ G T (C/T) (A/G) A C } 	ext{3'}$. The sequence provided in the question, $	ext{3'} 	ext{ G T P y P u A C } 	ext{5'} 	ext{ / } 	ext{5'} 	ext{ C A P u P y T G } 	ext{3'}$, is a generic representation of this exact recognition site. Hind II is unique because its recognition site is degenerate, meaning it allows for variation in the two central bases (Py and Pu). This enzyme was historically significant as the very first restriction endonuclease discovered and characterized, and it is known to cut the DNA helix symmetrically in the middle of the recognition sequence, producing $	ext{blunt ends}$ (cuts between Py and Pu), which are fragments without single-stranded overhangs.

The incorrect option is 1: Bam HI

Bam HI is a well-known restriction endonuclease, but its recognition site is very different from the one provided. Bam HI recognizes the specific six-base pair sequence $	ext{5'} 	ext{ G G A T C C } 	ext{3'}$ (and its palindrome, $	ext{3'} 	ext{ C C T A G G } 	ext{5'}$). Furthermore, Bam HI cuts off-center on this sequence between the two G bases, which results in $	ext{sticky ends}$ (cohesive ends) with a $	ext{4-base 5' overhang}$ ($	ext{G A T C}$). Since the recognition site in the question includes the degenerate bases $	ext{Py}$ and $	ext{Pu}$ and does not match the strict $	ext{GGATCC}$ sequence, it cannot be the restriction site for Bam HI.

The incorrect option is 2: Eco RI

Eco RI is one of the most commonly used restriction enzymes in molecular cloning, but its recognition site is $	ext{5'} 	ext{ G A A T T C } 	ext{3'}$. This sequence is a $	ext{palindromic sequence}$ where reading $	ext{5'} 	ext{ to } 	ext{3'}$ is the same on both strands. Eco RI also produces $	ext{sticky ends}$, cutting between $	ext{G}$ and $	ext{A}$ to leave a $	ext{4-base 5' overhang}$ ($	ext{A A T T}$). The given restriction site, $	ext{3'} 	ext{ G T P y P u A C } 	ext{5'} 	ext{ / } 	ext{5'} 	ext{ C A P u P y T G } 	ext{3'}$, does not contain the core $	ext{A A T T}$ sequence, nor does it match the highly specific, non-degenerate $	ext{G A A T T C}$ recognition sequence required by Eco RI.

The incorrect option is 4: Hind III

Hind III is closely related to Hind II in its nomenclature but recognizes a distinct sequence. The Hind III recognition sequence is the strict $	ext{palindromic sequence } 	ext{5'} 	ext{ A A G C T T } 	ext{3'}$ ($	ext{3'} 	ext{ T T C G A A } 	ext{5'}$). Like Bam HI and Eco RI, Hind III is known for producing $	ext{sticky ends}$ because it cuts asymmetrically between the two $	ext{A}$ bases at the $	ext{5'}$ end, leaving a $	ext{4-base 5' overhang}$ ($	ext{A G C T}$). The restriction site in the question is a $	ext{6-base}$ sequence that starts with $	ext{G T}$ on the $	ext{3'}$ end and ends with $	ext{A C}$ on the $	ext{5'}$ end of one strand, which clearly rules out the $	ext{A A G C T T}$ site specific to Hind III.