DNA replication produces two identical double helices from one original. The key experiment (Meselson-Stahl, 1958) showed that replication is semiconservative - each daughter molecule contains one original strand and one newly synthesized strand.
The replication fork, enzyme by enzyme
DNA replication
1
Scroll sideways to see the whole map.
Leading strand: continuous Lagging strand: in fragments RNA primer Parent strands
Why there is a lagging strand at allDNA polymerase can only add to a 3' end, so it can only build 5' to 3'. The two template strands run in opposite directions, so as the fork opens, one template presents its 3' end continuously and the other keeps presenting new 5' ends. The second one has to be copied in short backwards pieces.
Two different exonucleasesDNA polymerase III proofreads with 3'→5' exonuclease, removing a base it just added wrongly. DNA polymerase I uses 5'→3' exonuclease to chew forwards through the RNA primers and replace them. Same word, opposite directions, different jobs.
The end-replication problemThe very last primer on the lagging strand cannot be replaced, because there is no upstream 3' end to extend from. Chromosomes therefore shorten every division, which is what telomeres buffer and what telomerase reverses in stem cells and in most cancers.
Semi-conservative, bidirectional, and always 5' to 3'. Those three constraints between them force everything else on this map: the primers, the fragments, the two polymerases, the ligase, and the fact that chromosome ends are a problem at all.
The Replication Fork
Replication begins at an origin of replication. The double helix is unwound, creating a Y-shaped replication fork. DNA polymerase synthesizes new DNA on both template strands - but each strand is made differently because of the antiparallel geometry.
Leading strand: synthesized continuously 5’ to 3’ toward the fork.
Lagging strand: synthesized discontinuously in short 5’ to 3’ fragments called Okazaki fragments. These are later joined together.
Key Enzymes
| Enzyme | Job |
|--------|-----|
| Helicase | Unwinds the double helix at the fork |
| Topoisomerase (gyrase in bacteria) | Relieves supercoiling ahead of the fork |
| Single-strand binding proteins (SSBs) | Prevent reannealing of the separated strands |
| Primase | Synthesizes short RNA primers (~10 nt) that DNA polymerase can extend |
| DNA polymerase III (bacteria) / δ, ε (eukaryotes) | Main replicative polymerase. Adds dNTPs to the 3’-OH of the growing strand |
| DNA polymerase I (bacteria) | Removes RNA primers, fills in gaps with DNA |
| DNA ligase | Seals nicks between Okazaki fragments |
Fidelity
DNA polymerases have very low error rates - about one mistake per 107 bases. Several mechanisms contribute:
Base selection: the active site is shaped to fit correct base pairs much better than mismatches.
Proofreading: most replicative polymerases have 3’ to 5’ exonuclease activity that removes a mispaired base just after it is added, then tries again.
Mismatch repair: post-replication, a separate system scans newly made DNA for any surviving mismatches and corrects them (see DNA repair section).
Together these systems bring the overall error rate down to about one mistake per 109 bases.
What is semiconservative DNA replication, and how was it shown experimentally?
Click to reveal answer
Each daughter DNA molecule consists of one original (“parental”) strand and one newly synthesized (“daughter”) strand. Meselson and Stahl grew E. coli in 15N (heavy nitrogen), switched to 14N (light nitrogen), and tracked the DNA density. After one round of replication, all DNA had intermediate density (one heavy strand + one light strand), consistent only with semiconservative replication.
Why must the lagging strand be synthesized discontinuously?
Click to reveal answer
DNA polymerase only adds nucleotides to a free 3’-OH, meaning it synthesizes 5’ to 3’. At the replication fork, one template is oriented so synthesis runs into the fork (continuous leading strand). The other template is oriented so 5’ to 3’ synthesis would run AWAY from the fork. The lagging strand must therefore be synthesized in short fragments (Okazaki fragments), each started with a new primer as more template is exposed. Ligase later joins them.
Why are RNA primers needed at the start of each new DNA fragment?
Click to reveal answer
DNA polymerases cannot start a new strand from scratch - they can only extend an existing 3’-OH. Primase, a specialized RNA polymerase, does not need a primer and lays down a short RNA primer on the template. DNA polymerase then extends that primer. After replication, the RNA primers are removed and replaced with DNA (by polymerase I in bacteria, or by a separate enzyme in eukaryotes).
