DNA Replication

Semiconservative copying

Each strand of the parent helix templates a new partner, so every daughter molecule has one old strand and one new one: semiconservative replication ¹. Because the strands are complementary, either one fully specifies the other - the information is stored twice.

Origins and forks

Replication starts at origins, where initiator proteins open the helix ¹. Bacteria usually have one origin per chromosome; human chromosomes have thousands, so the genome can be copied in hours. From each origin two replication forks move in opposite directions, opening a growing bubble.

The crew at the fork

What DNA polymerase needs

DNA polymerase has four non-negotiable requirements ²:

1. a template to read;
2. a primer with a free 3’-OH - it can’t start a strand on its own;
3. the four dNTPs;
4. it only adds to the 3’ end, so synthesis runs 5’ to 3’.

Keep these in mind: they are the shopping list for PCR.

Leading and lagging strands

The templates are antiparallel and polymerase only goes 5’→3’, so the two new strands are made differently ¹:

• The leading strand is made continuously toward the fork from a single primer.
• The lagging strand is made away from the fork in short Okazaki fragments, each needing its own RNA primer. The primers are later removed and filled with DNA, and ligase joins the fragments.

Fidelity and proofreading

Two layers keep copying accurate ²:

• Base selection: correct pairs fit the polymerase’s active site far better than wrong ones.
• Proofreading: many polymerases have a 3’→5’ exonuclease that backs up, clips off a mismatched nucleotide, and resumes.

Proofreading cuts errors by orders of magnitude, and mismatch repair catches most of the rest ¹. In the lab this matters: Taq polymerase has no proofreading, while high-fidelity enzymes (Pfu and engineered relatives) do, and are chosen for cloning and sequencing templates.

Telomeres and telomerase

Linear chromosomes can’t be fully copied at their ends: when the last lagging-strand primer is removed there’s no 3’ end upstream to fill the gap, so each round shortens the chromosome - the end-replication problem ¹.

Telomerase carries its own RNA template and extends the repetitive telomeres (TTAGGG repeats in humans) to compensate. Stem and germ cells keep telomerase active; most somatic cells have little, so their telomeres shorten with age. Most cancers reactivate it.

The recipe for PCR

To copy DNA in a tube, supply exactly what polymerase demands ³:

Melt, anneal primers, extend, repeat: each cycle doubles the region between the primers. Amplification is covered in its own course.

What to take from this

Replication is semiconservative and starts at origins, with forks run by helicase, SSBs, topoisomerase, primase, polymerase, and ligase. Polymerase needs a template, a primed 3’-OH, dNTPs, and works only 5’→3’, so the leading strand is continuous and the lagging strand is built from Okazaki fragments. A 3’→5’ exonuclease proofreads (Taq lacks it). Telomerase rebuilds chromosome ends. PCR is replication stripped to polymerase’s requirements, with heat in place of helicase.