DNA Structure Part 2: Base Pairing, Chargaff's Rules, and X-ray Diffraction

Structural Attributes of the DNA Double Helix

• Foundational Model: The 1953 model proposed by James Watson and Francis Crick was based on earlier scientific contributions. This discussion focuses specifically on the chemical organization and physical evidence supporting that model.

• Ladder Analogy:

  • The structure of DNA is often depicted as a ladder.

  • Side Rails: The sugar-phosphate backbones represent the sides of the ladder.

  • Rungs: The nitrogenous bases represent the interior steps or rungs of the ladder.

• Antiparallel Orientation:

  • The two strands of the DNA molecule run in opposite directions, a configuration known as antiparallel.

  • One strand terminates at the $5'$ end while its partner terminates at the $3'$ end, and vice versa.

Chemical Properties and Bonding

• Complementary Base Pairing:

  • The nitrogenous bases on opposite strands pair in a specific, complementary manner.

  • Thymine ($T$) always pairs with Adenine ($A$).

  • Guanine ($G$) always pairs with Cytosine ($C$).

  • A pyrimidine (single-ring structure) always pairs with a purine (double-ring structure).

• Hydrogen Bonding:

  • The connection between nitrogenous bases consists of hydrogen bonds, which are considered weak bonds in a biological context.

  • Specific Bond Counts:

    • Between Cytosine ($C$) and Guanine ($G$), there are $3$ hydrogen bonds.

    • Between Adenine ($A$) and Thymine ($T$), there are $2$ hydrogen bonds.

  • Structural Stability: While individual hydrogen bonds are weak, the vast quantity of these bonds throughout the DNA molecule makes the overall structure highly stable.

  • Replication Implications: It is functionally important that these bonds are easily broken (as opposed to strong covalent bonds) so the two strands can separate for the purpose of DNA replication.

Directionality and the Significance of 5' and 3' Ends

• Terminal Chemical Entities:

  • Each end of a DNA strand is distinguished by a specific chemical group attached to the deoxyribose sugar's carbons.

  • The $5'$ (Five Prime) End: Characterized by a free phosphate group attached to the five-prime carbon ($C5$).

  • The $3'$ (Three Prime) End: Characterized by a free hydroxyl group ($OH$) attached to the three-prime carbon ($C3$).

• DNA Polymerase Function:

  • DNA polymerase is the enzyme responsible for synthesizing more DNA during replication.

  • Growth Direction: The enzyme synthesizes DNA in the $5' \rightarrow 3'$ direction.

  • Nucleotide Addition: New nucleotides are added exclusively to the $3'$ end. Consequently, the $3'$ end is referred to as the growing end of the DNA molecule.

  • Reference Convention: In academic and scientific discourse, DNA sequences are always discussed or read in the $5'$ to $3'$ direction.

Chargaff’s Rules of Base Composition (1948)

• Research Overview: In 1948, Erwin Chargaff published findings on the relative amounts of the four nitrogenous bases ($A, T, G, C$) found in the DNA of various organisms.

• Empirical Findings by Organism:

  • E. coli: Found to have roughly equal amounts of all four bases.

  • Yeast:

    • Adenine ($A$): $31.3\%$

    • Thymine ($T$): $32.9\%$

    • Guanine ($G$): $18.7\%$

    • Cytosine ($C$): $17.1\%$

  • Human Liver: Showed a similar pattern of roughly equal percentages for $A/T$ and $G/C$, with Adenine specifically measured at $30.3\%$.

  • Rat Bone Marrow: Used to verify the relationship between total purines and total pyrimidines.

• The Empirical Rules:

  • Rule 1: The total amount of Adenine equals the total amount of Thymine ($A = T$), and the total amount of Cytosine equals the total amount of Guanine ($C = G$).

  • Rule 2: The total amount of purines ($A + G$) always equals the total amount of pyrimidines ($T + C$).

  • These rules allow scientists to determine the proportions of all four bases if the percentage of even one base is known.

X-Ray Diffraction and the Contribution of Rosalind Franklin

• Photograph 51: This specific X-ray diffraction image was captured by the scientist Rosalind Franklin while she was working in England.

• Evidence Provided: The photograph provided the physical proof that DNA was:

  1. Helical in nature.

  2. Double-stranded.

  3. Antiparallel.

• Historical Context and Controversy:

  • Maurice Wilkins, a scientist at the same university, took Photograph 51 without Franklin's knowledge and showed it to Watson and Crick (who were at a different university).

  • Watson and Crick identified this image as the "moment" that clarified the physical structure of the DNA model they were building.

  • Rosalind Franklin published her findings in a paper back-to-back with Watson and Crick's 1953 publication.

  • She did not receive a Nobel Prize because she passed away from ovarian cancer before the prize was awarded.

  • Recommended Reading: The Dark Lady of DNA by Brenda Maddox is suggested for a more detailed account of her life.

Quantitative Analysis and Sample Calculation

• Scenario: A sample of DNA from an organism contains $31\%$ Guanine ($G$).

• Identify Thymine ($T$) Percentage:

  1. According to Chargaff's rules, if $G = 31\%$, then Cytosine ($C$) must also be $31\%$.

  2. Calculate the combined total for $G$ and $C$:        $31\% + 31\% = 62\%$.

  3. Subtract this total from the full $100\%$ of the sample to find the remainder for $A$ and $T$:        $100\% - 62\% = 38\%$.

  4. Because $A = T$, the $38\%$ must be divided equally between the two:        $\frac{38\%}{2} = 19\%$.

• Result: The percentage of Thymine in the sample is $19\%$.