Question

What part(s) of a nucleotide will occupy the major & minor groove of a double-stranded DNA molecule? WHY? What parts are found in the DNA backbone? WHY? Where would a Restriction Endonuclease associate with DNA? How long is a double-stranded DNA molecule that is 2 x 105 bp? How many nucleosomes would be required to package this DNA if it were in a Eukaryote? When chromatin from any eukaryote is digested briefly with micrococcal nuclease (an endonuclease) and fractionated using electrophoresis, DNA fragments of approximately 200 base pairs in length are observed. Explain this result. When preparing the above-mentioned chromatin, you were distracted and allowed the micrococcal nuclease digestion to proceed for a longer time. Does this extended period of digestion change your results? If so, explain how. If not, explain why.

Answer 1

The nitogenous bases of the nucleotide occupy the major and minor grooves of the DNA helix. They are so located as they are hydrophobic in nature. Being inside the groove facilitates interaction between the nitrogenous groups and protects them as they are hydrophobic in nature. In addition this favors hydrogen bonding between the bases, stabilizing the DNA double helix.

The phosphate groups and sugar moiety is found in the backbone. The phosphate group gives an overall negative charge to the DNA making it hydrophilic. This favors interaction with the surrounding water molecules. The phosphate group and sugar moiety helps to maintain the DNA in a hydrophilic environment.

Restriction endonuclease binds to the major and minor grooves of DNA and interact with the bases that gives them sequence specificity.

The distance between two base pairs in DNA is 0.34 X 10^{^-9} m.

length of DNA= No.of bp x 0.34 X 10^-9 m

2 x10⁵ X 0.34 X 10^{^-9} m = 6.8 x10^-5 m =0.068 mm

One nucleosome wraps around 150 bp of DNA. So the number nucleosome required to pack 2 x 10⁵ bp will be = 2 x 10⁵ bp/150 bp= 1333.33. Approximately 1334 nucleosomes required to pack 2 x 10⁵ bp DNA.

Micrococcal nuclease preferentially digest in the inter nucleosomal linker region of the chromatin. Since each nucleosome wraps around 150 to 200 bp of DNA the linker region that MNase recognize is situated every 200 bp. Thus controlled digestion with MNase and subsequent electrophoresis yields 200 bp (and its multiple) length DNA fragments. Thus electrophoresis shows multiple bands representinf mononucleosomes (200 bp), dinucleosomes (400 bp), trinucleosomes (600 bp) and undigested chromatin.

Longer period of digestion will alter the result obtained. Long period of digestion leads to maximal digestion of the chromatin and all the linker regions present in the chromatin are cleaved thus generating only mononucleosomes of 200 bp each that can be observed in the agarose gel.