DNA replication is the process by which a cell duplicates its genome before cell division, ensuring each daughter cell receives an identical copy of the genetic material. The process is semiconservative, meaning each new DNA molecule consists of one original strand and one newly synthesized strand. This principle is also the basis for PCR, a technique used to amplify specific DNA sequences.
Origins of Replication
Replication begins at specific sequences called origins of replication. In E. coli, the single origin is called oriC, a 245-base-pair region containing DnaA box sequences and AT-rich repeats. The initiator protein DnaA binds to these sequences, causing local melting of the AT-rich region. Eukaryotic chromosomes contain multiple origins of replication spaced approximately every 30 to 100 kilobases. The licensing system ensures each origin fires only once per cell cycle by loading the Mcm helicase complex during G1 phase.
The Replication Fork
Once the DNA is opened, the replication machinery assembles at the replication fork. DNA helicase unwinds the double helix, with DnaB in bacteria and the CMG complex containing Mcm2-7 in eukaryotes. Single-strand binding proteins coat the exposed strands, preventing reannealing and protecting them from nucleases. Topoisomerase relieves the torsional stress created by unwinding ahead of the fork.
DNA Polymerases
DNA polymerases catalyze the addition of nucleotides to the 3-prime end of a growing DNA strand, requiring a template and a primer with a free 3-prime hydroxyl. They always synthesize in the 5-prime to 3-prime direction and cannot initiate synthesis de novo. E. coli has five DNA polymerases, with Pol III being the main replicative enzyme. Eukaryotes have at least 15, with Pol delta and Pol epsilon responsible for lagging and leading strand synthesis respectively.
Leading and Lagging Strand Synthesis
Because DNA polymerases can only synthesize in the 5-prime to 3-prime direction, the two strands are replicated differently. The leading strand is synthesized continuously in the same direction as fork movement, requiring only one primer. The lagging strand is synthesized discontinuously in short fragments called Okazaki fragments, each requiring a new primer. In eukaryotes, Okazaki fragments are about 100 to 200 nucleotides long. Primase, a specialized RNA polymerase, synthesizes short RNA primers of about 10 nucleotides.
Primer Removal and Ligation
The RNA primers on the lagging strand must be removed and replaced with DNA. In bacteria, DNA polymerase I removes the RNA primer through its 5-prime to 3-prime exonuclease activity and fills the gap. In eukaryotes, RNase H removes most of the primer, and FEN1 nuclease removes the last ribonucleotide. DNA ligase seals the nick between adjacent fragments, consuming NAD+ in bacteria or ATP in eukaryotes and archaea. Restriction enzyme digestion generates compatible fragments that can be joined by DNA ligase in molecular cloning.
Replication Fidelity
DNA replication achieves remarkable accuracy, with error rates of about one mistake per 10^10 nucleotides. This fidelity results from three mechanisms. First, DNA polymerases have high geometric selectivity, discriminating against incorrect base pairing during incorporation. Second, the 3-prime to 5-prime exonuclease activity of replicative polymerases proofreads newly added bases, removing mismatches immediately. Third, post-replication mismatch repair corrects errors that escape proofreading. Defects in mismatch repair cause microsatellite instability and predispose to hereditary non-polyposis colorectal cancer.
Telomere Replication
The ends of linear eukaryotic chromosomes pose a special problem because DNA polymerase cannot replicate the very end of the lagging strand. Telomeres, repetitive DNA sequences at chromosome ends, are extended by telomerase, a specialized reverse transcriptase that carries its own RNA template. Telomerase adds TTAGGG repeats in humans, preventing progressive shortening. Telomerase is active in germ cells, stem cells, and most cancer cells, while somatic cells lack telomerase and undergo telomere shortening with each division, contributing to cellular senescence.
