DNA (deoxyribonucleic acid), which carries genetic information, has a double-helix structure. Before cell division, it is accurately replicated so that identical information is passed to daughter cells.
Basic Knowledge
Chemical structure of DNA: Two polynucleotide strands, each consisting of nucleotides (base+deoxyribose+phosphate) linked by phosphodiester bonds in the 3'→5' direction, face each other in an antiparallel arrangement and form a double helix through hydrogen bonds between bases. Bases: A (adenine), T (thymine), G (guanine), and C (cytosine). There are two hydrogen bonds between A-T and three hydrogen bonds between G-C. Chargaff’s rule: A=T and G=C.
Watson-Crick model (1953): The double-helix structure was proposed from X-ray crystallographic data (Franklin) and Chargaff’s rule. The helix has a diameter of 2 nm, a pitch (one turn) of 3.4 nm, and a distance between base pairs of 0.34 nm.
DNA replication (semiconservative replication): ① Helicase unwinds the double strand and forms a replication fork. ② DNA polymerase reads the template strand in the 3'→5' direction and synthesizes a complementary new strand in the 5'→3' direction. ③ Because DNA polymerase requires an existing 3' end, primase first synthesizes an RNA primer, after which extension begins. ④ The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously as Okazaki fragments, which are joined by DNA ligase. ⑤ The Meselson-Stahl experiment (1958) demonstrated semiconservative replication (¹⁵N/¹⁴N labeling).
Explain the evidence from the Meselson-Stahl experiment that proved semiconservative DNA replication.
Answer: Escherichia coli grown in a ¹⁵N medium (all DNA was the heavy ¹⁵N-¹⁵N form) was transferred to a ¹⁴N medium and cultured for one generation. Density-gradient centrifugation showed DNA as one band of intermediate density, the ¹⁵N-¹⁴N form. If replication had been conservative, two bands (heavy+light) would have appeared. This result provided evidence for semiconservative replication.
- Double helix = antiparallel; A-T (2 hydrogen bonds); G-C (3 hydrogen bonds)
- Chargaff’s rule = A=T and G=C
- Semiconservative replication = one old strand+one new strand in daughter DNA
- DNA polymerase = synthesizes only in the 5'→3' direction; requires a primer
- Helicase = unwinds the double strand and forms a replication fork
- Okazaki fragments = units of discontinuous synthesis on the lagging strand
- Meselson-Stahl experiment = demonstration of semiconservative replication
Points to Note
① DNA polymerase can synthesize only in the 5'→3' direction and reads the template strand in the 3'→5' direction. ② The RNA primer is required for DNA polymerase to begin extension; it is later removed by RNaseH and replaced with DNA. ③ G-C base pairs have three hydrogen bonds and are stronger than A-T pairs, so DNA with a high G+C content has a higher melting temperature (Tm).
Practice
- State the numbers of hydrogen bonds between A-T and G-C in the DNA double helix.
- Explain why the lagging strand is synthesized discontinuously during semiconservative DNA replication.
- Name the two nitrogen isotopes used in the Meselson-Stahl experiment and describe the experimental results.