DNA structure

A. Deoxyribonucleotides: the monomeric units

Deoxyribonucleotide monophosphate

1. Purines

2. Pyrimidines

B. Primary structure: the linear chain

1. 5’ and 3’ ends (5’-NNNNN-3’ or 5’-pNpNpNpNpN-3’)

2. Phosphodiester bond

C. Secondary/quaternary structure

1. The dreaded double helix *

2. Base pairing

a) Watson and Crick (1953) based their base pairing on the work of Chargaff. ²

* WATSON, J. D., & F. H. C. CRICK. Molecular structure of nucleic acids: a structure for deoxyribosenucleic acid. Nature 171(4356): 737–738, 1953.

2 CHARGAFF, ERWIN. Chemical specificity of nucleic acids and mechanism of their enzymatic degradation.Experientia 6: 201–209, 1950

3. Antiparallel construction

4. Major and minor grooves

D. Tertiary structure: supercoiling

1. The bacterial nucleoid: a relatively ribosome-free portion of the cell thought to contain most of the DNA

a) Not separated from the cytoplasm by a membrane: one ofthe hallmarks of the prokaryotic cell

2. Supercoiling in the nucleoid

a) The DNA is compacted some 1000-fold in the linear dimension (1 mm of DNA in a 1-μm cell).

b) Supercoiling compacts the DNA in the linear dimension.

(1) Most DNA in bacteria is negatively supercoiled, with an average of one supercoil for every 300 bp

c) Topoisomerases catalyze the introduction and removal of supercoils

(1) Type I topoisomerases nick the DNA (cut one strand) to introduce or relieve supercoiling one supercoil at a time

(a) e.g. topA encodes the TopA protein; topA mutants are more negatively supercoiled than usual, so TopA relieves supercoiling

(b) topA mutants grow more slowly, but still grow

(2) Type II topoisomerases cut both strands to introduce or relieve supercoiling two supercoils at a time

(a) E. coli’s DNA gyrase, a type II enzyme, is encoded by gyrA and gyrB. The active enzyme consists of two GyrA polypeptides and two GyrB polypeptides.

(i) GyrA hydrolyzes the DNA and holds it.

(ii) GyrB has the binding site for ATP, which provides the energy for twisting.

(iii) DNA gyrase is used to introduce negative

supercoiling.

(iv) Inhibition of DNA gyrase “will cause cell death.” gyrA or gyrB mutants do not grow