4.1 DNA Structure, Replication, and Repair
Key Takeaways
- DNA is a double helix of two antiparallel strands held together by complementary base pairs: A-T (two hydrogen bonds) and G-C (three hydrogen bonds).
- Replication is semiconservative: each daughter molecule contains one parental strand and one newly synthesized strand, as proven by the Meselson-Stahl experiment.
- DNA polymerase III adds nucleotides only 5' to 3', so the leading strand is synthesized continuously while the lagging strand is built in short Okazaki fragments later joined by DNA ligase.
- Telomerase extends the 3' ends of linear chromosomes using an RNA template, offsetting the end-replication problem in germline and stem cells.
- Three major repair pathways protect the genome: mismatch repair (post-replication errors), nucleotide excision repair (bulky/UV damage), and base excision repair (single damaged bases).
Why DNA Replication Matters for the Praxis
The Praxis Biology (5235) test rewards candidates who can connect molecular structure to enzyme function. Replication questions typically ask you to identify an enzyme by its job, predict the consequence of a mutation in one of those enzymes, or interpret a diagram of a replication fork. To answer them quickly, you need a precise vocabulary, not vague memorization.
The Double Helix
In 1953, James Watson and Francis Crick proposed that DNA is a right-handed double helix of two antiparallel strands. One strand runs 5' to 3', the other runs 3' to 5'. The sugar-phosphate backbone faces outward; the nitrogenous bases face inward and pair through hydrogen bonds.
| Base Pair | Hydrogen Bonds | Category |
|---|---|---|
| A - T | 2 | Purine - Pyrimidine |
| G - C | 3 | Purine - Pyrimidine |
The extra hydrogen bond in G-C pairs makes GC-rich regions more thermally stable, which is why PCR primers near GC-rich templates need higher annealing temperatures.
Chargaff's Rules
Before Watson and Crick, Erwin Chargaff found that in any double-stranded DNA sample, %A = %T and %G = %C. If a genome is 30% adenine, it must be 30% thymine, leaving 40% for G and C combined (20% G and 20% C).
Semiconservative Replication
Replication is semiconservative: each daughter molecule contains one parental strand and one newly synthesized strand. This was proven by the 1958 Meselson-Stahl experiment, which grew E. coli in heavy (15N) nitrogen, then shifted them to light (14N) nitrogen and used density-gradient centrifugation to show a single hybrid band after one round of replication.
The Replication Fork
Replication starts at an origin of replication (multiple origins in eukaryotes, one in prokaryotes). Each origin produces two replication forks moving in opposite directions.
| Enzyme/Protein | Job |
|---|---|
| Helicase | Unwinds the parental double helix at the fork |
| Single-strand binding proteins (SSB) | Stabilize separated single strands |
| Topoisomerase | Relieves supercoiling ahead of the fork by cutting and rejoining strands |
| Primase | Synthesizes a short RNA primer that DNA polymerase can extend |
| DNA polymerase III | Main replicative polymerase in prokaryotes; adds DNA nucleotides 5' to 3' |
| DNA polymerase I | Removes RNA primers and replaces them with DNA |
| DNA ligase | Seals the phosphodiester backbone between adjacent Okazaki fragments |
Because DNA polymerases only add to a free 3'-OH, synthesis is always 5' to 3'. That single rule explains the asymmetry of the fork.
Leading vs. Lagging Strand
- The leading strand runs 3' to 5' on the template, so the new strand is built 5' to 3' continuously toward the fork after a single RNA primer.
- The lagging strand template runs 5' to 3', so the new strand must be built away from the fork in short Okazaki fragments (≈ 1,000-2,000 nt in bacteria, ≈ 100-200 nt in eukaryotes), each started by its own primer and later joined by DNA ligase.
Telomeres and Telomerase
Linear eukaryotic chromosomes face an end-replication problem: after each round, the lagging strand cannot fully replicate the 3' end of the template, so chromosomes shorten. Telomeres are repetitive non-coding caps (TTAGGG in humans) that buffer this loss. Telomerase, a reverse-transcriptase ribonucleoprotein, extends the 3' overhang using its own RNA template. Telomerase is active in germline, stem, and most cancer cells, but largely off in differentiated somatic cells.
DNA Repair Pathways
Replication errors and environmental damage create lesions that must be fixed before they become permanent mutations.
- Mismatch repair (MMR) corrects base-pair mismatches that escape polymerase proofreading. MutS recognizes the mismatch; MutL and MutH excise the newly synthesized strand and DNA polymerase resynthesizes it. Loss of MMR causes Lynch syndrome (hereditary colorectal cancer).
- Nucleotide excision repair (NER) removes bulky, helix-distorting lesions such as UV-induced thymine dimers. A short oligonucleotide containing the lesion is cut out and replaced. Defective NER causes xeroderma pigmentosum.
- Base excision repair (BER) removes single damaged bases (deaminated, oxidized, or alkylated). A DNA glycosylase removes the base, AP endonuclease nicks the backbone, and a polymerase plus ligase complete the patch.
A quick mnemonic: MMR fixes wrong base, NER fixes bulky damage, BER fixes a single damaged base.
A student examines a replication fork and identifies a strand that is being synthesized in short, discontinuous fragments away from the direction of fork movement. Which combination of enzymes is most directly responsible for completing this strand?
A patient has xeroderma pigmentosum, an inherited inability to repair UV-induced thymine dimers in skin DNA. Which DNA repair pathway is most likely defective?