Hydrolysis

This is the breaking of bonds by the addition of water. The DNA double helix can be broken by either of two methods, chemical or enzymatic hydrolysis.

Chemical Hydrolysis

DNA is stable to bases (alkaline hydrolysis). This is one of the features which distinguish DNA from RNA (RNA is not stable under alkaline conditions).

Strong acids at a high temperature (e.g. 6M HCl at 110^oC for 18 hours), are capable of breaking the DNA molecule into its components. These conditions break both of the phosphate ester bonds and also the N-glycosidic bond between the deoxyribose and the purines and pyrimidine bases. The products of this mixture are the 4 bases, phosphoric acid, and deoxyribose which then polymerises to produce a brown sticky tar.

The bases resulting from acid hydrolysis can be separated by paper chromatography, and then quantified. This means the percentage composition of the bases in the DNA strand can be calculated.

Enzymatic Hydrolysis

This is performed by enzymes called "nucleases". Some of these enzymes can carry out similar reactions on RNA.

EXONUCLEASES
These proteins cleave nucleotides from the ends of the DNA molecule. There are 5' exonucleases (cleave the DNA from the 5'-end of the DNA chain) and 3' exonucleases (cleave the DNA from the 3'-end of the chain).

Different exonucleases can hydrolyse single-strand or double strand DNA. (Remember the exonuclease activities of DNA polymerase.)

ENDONUCLEASES
The endonucleases have the capablity to cleave the bonds within a DNA strand. Endonucleases may also be specific for either DS-DNA or SS-DNA. Endonuleases can either cleave randomly within the DNA or can cleave at specific sequences. Endonucleases which cleave double strand DNA at random often make interactions with the backbone of DNA. This can be seen in the close proximity of certain amino acids (polar amino acids) around the DNA in the following image. . This image is of a deoxyribonuclease called DNAaseI which cleaves bonds in double strand DNA more or less at random. The products of the reaction with DNA are oligo/polynucleotides.

The interaction of the polar amino acid side chains (green) with the backbone groups of the DNA can be seen more clearly if the rotation function is started. The phosphorous atoms of the DNA backbone are the gold spheres.

Restriction Endonucleases
These are endonucleases which recognise specific sequences within double-strand DNA and cleave both strands either within or close to the recognition sequence. You will hear a lot more about them in later sections of the course when the topic of recombinant DNA manipulations is covered.

For instance, EcoRV (A restriction endonuclease found in E.coli) recognises the sequence below, and cleaves at the arrows...

The image in the frame to the left shows the backbone of the enzyme protein in blue. The enzyme actually is a dimer of two identical subunits which are both shown in the image. The DNA substrate bound to the enzyme contains the sequence of base pairs recognised by EcoRV. The enzyme is thought to bind initially in a non sequence-specific manner to the DNA by interactions with the deoxyribose-phosphate backbone and move along the double helix until it reaches a target site.

When this occurs some of the amino acid side chains of the protein which are close to the DNA make specific contacts with the sides of the base-pairs in the major and minor grooves of the DNA. These are shown in green in the image. If the "rotation" function is turned on then the way in which these side chains interact in the grooves of the DNA can be seen. (The backbone of the DNA strands is marked by the yellow phosphorous atoms.)

As the molecule rotates you can see that there are some side chains which are close to the backbone atoms. These are the groups responsible for the non-specific interactions by which the enzyme initially interacts with the DNA.

The Supercoil

Some DNA molecules are circular,with the two strands being covalently closed circles in the double helix, for instance:

 

	Most bacterial and many eukaryotic viruses.
	Chromosomal DNA of most prokaryotes.
	Organelle (mitochondria & chloroplast) DNA of eukaryotes.

Relaxed DNA has 10 base pairs per turn of the helix (see previous tutorial if you are unsure). If the DNA helix is wound tighter/looser, it has more/less base pairs per turn. This induces some strain on the molecule, which can be relieved by partial separation of the strands or by the helix twisting around itself in a supercoil.

The native state of DNA is negatively supercoiled (unwound, with fewer turns than expected for the number of base pairs). Positively supercoiled DNA is overwound with more turns than expected for the number of base pairs. The phenomenon of supercoiling also applies to eukaryotic chromosomes. Although the DNA in eukaryotic chromosomes is linear it is fixed to a protein matrix every so often, resulting in DNA loops where the ends of the DNA is fixed and not free to rotate. This allows the DNA to be supercoiled, even though it is linear.

So now you know something about the interactions which maintain the DNA in the double helix and how these interactions may be broken by relatively small changes in the environment of the DNA. Breaking the primary structure of DNA requires quite severe chemical conditions but can be accomplished in vivoquite readily by enzymes.

Move on to find out how some proteins which bind to DNA can bring about separation of the two strands to allow access to the genetic code for DNA replication and for expression of the genes. Pust the NEXT button, or refresh your memory of the helix structure by pressing BACK if you feel you need to revisit this.