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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

The Hershey and Chase experiment was one of the leading suggestions that DNA was a genetic material. Hershey and Chase used phages, or viruses, to implant their own DNA into a bacterium. They did two experiments marking either the DNA in the phage with a radioactive phosphorus or the protein of the phage with radioactive sulfur. With the bacteria that was infected by the phages with radioactive DNA the DNA in the bacteria was radioactive. In the bacteria that was infected with the radioactive protein the bacteria was radioactive, not the DNA. This proves that DNA is a genetic material and it is passed on in viruses.

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

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.

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