General Biology/Genetics/DNA, The Genetic Material

DNA

DNA stands for Deoxyribose Nucleic Acid. That is, a nucleic acid with two sugars. DNA is the hereditary material of cells and is considered the blueprint of life. DNA is found in all kingdoms of life. Even most viruses have DNA. A molecule of DNA is chemically stable (it does not have a 2-prime alcohol group.)

When someone says DNA, they may be referring to one's genetic material on multiple levels: They may be speaking about a single deoxyribose nucleic acid molecule, a section of a double helix, a section of a chromosome, or one's entire hereditary composition.

antiparallel
Double helix
- Semiconservative replication
- Sequence of nucleotides encodes functional RNA or polypeptide

Historical perspective

Mitosis and meiosis
- Regular distribution of chromosomes suggested that they contain hereditary information
- Bridges/Morgan, using Drosophila melanogaster showed that genes are on chromosomes (1910s)
Hammerling: nucleus contains hereditary information (1930s)
Griffith: transformation of bacteria (1928)
Avery, MacLeod, McCarty: transforming substance is DNA (1944)
Hershey, Chase: DNA is hereditary material of viruses (1952)
Rosalind Franklin
Watson and Crick: structure of DNA (1953)

Hershey-Chase Experiment

Two batches of isotopically labeled bacteriophage T2 particles were prepared. One was labeled with 32P in the phosphate groups of the DNA, the other with 35S in the sulfur-containing amino acids of the protein coats (capsids). (Note that DNA contains no sulfur and viral protein contains no phosphorus.) The two batches of labeled phage were then allowed to infect separate suspensions of unlabeled bacteria. Each suspension of phage-infected cells was agitated in a blender to shear the viral capsids from the bacteria. The bacteria and empty viral coats (called “ghosts”) were then separated by centrifugation. The cells infected with the 32P-labeled phage were found to contain 32P, indicating that the labeled viral DNA had entered the cells; the viral ghosts contained no radioactivity. The cells infected with 35S-labeled phage were found to have no radioactivity after blender treatment, but the viral ghosts contained 35S. Progeny virus particles (not shown) were produced in both batches of bacteria some time after the viral coats were removed, indicating that the genetic message for their replication had been introduced by viral DNA, not by viral protein.

DNA/RNA components

Miescher: discovered DNA, 1869

Structure of DNA

DNA is in a double helix structure made up of nucleotides. The "backbone" of the double helix is composed of phosphates connected to a five-carbon sugar called deoxyribose. The "rungs" are composed of organic compounds, purines and pyrimidines. Purines contain Adenine(A) and Guanine(G) and have two rings in their structures. Pyrimidines contain Cytosine(C) and Thymine (T) and have one ring in their structures.

Chemical structure of DNA

Polynucleotide
- Phosphodiester bonds between nucleotides
- 5’-pGpTpCpGpTpApApTp-OH 3’
Chargaff’s rules, in DNA: equimolar amounts
- A = T
- G = C

3D structure of DNA

James Watson and Francis Crick (1953)
- Nucleotide
- Keto and amino forms of bases
- Chargaff’s rules
- X-ray crystallographic data (Rosalind Franklin)

Franklin

X-ray diffraction of DNA crystals
revealed regular pattern explained by antiparallel double helix

DNA model

Double helix of polynucleotides
- antiparallel
- 3’-5’ phosphodiester bonds
Base pairs held by hydrogen bonds
- AT
- GC
There are about 10 base pairs per turn of helix
model has predictive power
- mode of DNA replication
- encoding of genetic information

DNA replication

Conservative model
- One double helix of both old strands
- One double helix of two new strands
Dispersed
- Each strand mixture of old new
Semiconservative
- Meselson-Stahl experiment confirmed its viability over the previous two
  - grew E. coli bacterium in a culture containing 15N (a heavy isotope of nitrogen)
  - bacterium assimilated the 15N into their DNA
  - a similar process was then done using 14N, a lighter isotope
  - following centrifugation, the densities were observed to be that of combined in the middle, and 14N on top, thereby confirming the semiconservative model

DNA replication

Semiconservative
New nucleotides added to 3’ –OH
Replication fork
- Replication complex
DNA polymerase
Associated enzymes/proteins
- Energy from phosphate bonds of triphosphate nucleotide substrates (dNTP)

DNA polymerases

Prokaryotes, E. coli
- 3 DNA polymerases
- III is main enzyme for DNA replication
- ~1000 nt/sec
Eukaryotes
- 6 DNA polymerases
Add nucleotide to 3’ –OH end
All require primer, i.e., free 3’ –OH

DNA replication complex

Helicase "unzips" the DNA double helix
Primase: synthesize RNA primer
Single-strand binding proteins
DNA gyrase (topoisomerase)
DNA polymerase III
DNA polymerase I (remove primer, fill gaps)

DNA replication

5’ → 3’ replication
- Nucleotide addition at 3’ –OH
- No exceptions
New strands are oriented in opposite direction due to 5’ → 3’ constraint
- Leading strand: continuous replication
- Lagging strand: discontinuous replication
  - contains multiple Okazaki fragments
Joined by DNA ligase

DNA replication fork

primer required by all DNA polymerases

Replication units

Replicon

A region of DNA that is replicated from a single origin.

What is gene?

Garrod
- “inborn errors of metabolism”
- Alkaptonuria: enzyme deficiency
Beadle and Tatum
- One gene one enzyme
- Genetic and biochemical analysis in Neurospora
Today: gene is sequence of nucleotides encoding functional RNA molecule or the amino acid sequence of a polypeptide

References

This text is based on notes very generously donated by Paul Doerder, Ph.D., of the Cleveland State University.