Quick questions restriction enzymes

[scilearner]

Hello everyone,

 

 

1. Restriction enzymes cut only sugar phospahte bonds right. So in the formation of sticky bonds for example in BamHl, how does the hydrogen bonds between the molecules break. When sugar phophate bonds on each strand break, does it pull on the free side dissociating the hydrogen bonds. Can these pieces stick back easily again or do they need a ligase?

 

2. Can restriction enzymes cut single stranded DNA?

 

3. Also how do these detect specific sequences of DNA. Do they have some complementary area that binds with the specific region.

 

Thanks

[MCB]

1. First it is important to realize that Hydrogen bonds are weak chemical bonds. When the DNA is in its double stranded helical form the complementary base pairs are basically held in a position for maximum hydrogen bonding to occur between them. When the phosphate backbone is broken, the nucleotides at the sticky ends are no longer physically held in place by the backbone and the weak hydrogen bonds can break (many hydrogen bonds in unison can be strong, but the small amount in the sticky overhangs of 4-10 base pairs is not very significant). The sticky ends of a double stranded DNA that has been cut by restriction enzymes can easily come back together to hydrogen bond too. In fact, when ligase is added to cloning reactions the number of DNA molecules that just re-ligate back together is much greater than those that actually receive the foreign DNA insert. Ligase is needed any time the phosphate backbone is broken.

 

2. I believe it is possible. For the most part though i think most restriction enzymes act as dimers and recognize palindromic DNA sequences to cut dsDNA.

 

3. Restriction enzymes and other sequence specific DNA binding proteins recognize the specific sequence through chemical interactions between the DNA and amino acid side chains in the DNA binding motif of the protein. Usually, the protein recognizes certain features in the Major groove of the DNA in which it can distinguish between every type of base pairing! really exciting stuff

 

There's really a LOT more to all of these things you're inquiring about but you'll learn all of that in a molecular genetics course if you so choose that path!

[Genecks]

Response to #2: Yes.

 

Nucleic Acids Res. 1985 August 26; 13(16): 5747–5760.

 

Type II restriction endonucleases cleave single-stranded DNAs in general.

K Nishigaki, Y Kaneko, H Wakuda, Y Husimi, and T Tanaka

 

Abstract

Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.

 

By the way, scilearner, if you don't mind me asking, ... what are you up to?

You seem to be specializing your reading in biological sciences at the moment.

 

Are you a student currently in an accredited educational institution or an autodidact?

Edited January 10, 2011 by Genecks