AP Syllabus focus:
‘DNA polymerase requires short RNA primers to initiate synthesis of new strands and extends DNA by adding nucleotides to the 3? end.’
DNA replication depends on enzymes that can start and extend new DNA strands accurately. This page focuses on why RNA primers are needed and how DNA polymerase builds DNA in a specific direction.
Why DNA polymerase needs an RNA primer
DNA polymerase cannot begin a new strand from nothing; it can only add nucleotides onto an existing nucleic acid strand that provides a free 3′ hydroxyl group.
RNA primer: A short RNA sequence that provides a free 3′-OH end for DNA polymerase to begin DNA synthesis.
The primer is made by primase (an RNA polymerase enzyme). Using the DNA template, primase synthesises a short, complementary RNA segment, creating the starting point needed for DNA polymerase to take over.
Key idea: “start” vs “extend”
Primase: starts a strand by making RNA (can begin de novo).
DNA polymerase: extends a strand by adding DNA nucleotides to the primer’s 3′ end.
How DNA polymerase extends DNA strands
DNA polymerase adds deoxyribonucleotides (dNTPs) one at a time to the 3′ end of the growing strand, forming phosphodiester bonds. Because addition only occurs at the 3′ end, synthesis proceeds 5′ → 3′ along the new DNA strand, while the enzyme reads the template strand 3′ → 5′.
Labeled replication-fork schematic showing that new DNA is synthesized only in the direction. It highlights how primase makes short RNA primers and how DNA polymerase extends from those primers continuously on the leading strand and discontinuously (Okazaki fragments) on the lagging strand. Source
DNA polymerase: An enzyme that synthesises DNA by adding dNTPs to the 3′ end of a pre-existing strand, using a DNA template and base-pairing rules.
This directionality matters for both accuracy and coordination: the enzyme’s active site is structured to catalyse bond formation only with a correctly paired incoming nucleotide at the 3′ end.
What DNA polymerase requires to work
A DNA template strand to copy
A primer-template junction (double-stranded region with a free 3′-OH)
dNTPs (A, T, G, C) as substrates for both building the strand and providing energy
Appropriate cellular conditions (ions such as Mg, enzyme cofactors)
Accuracy and proofreading (AP-level emphasis)
A major reason DNA replication is reliable is that many DNA polymerases have proofreading capability:
They check whether the newly added nucleotide is correctly base-paired.
If an incorrect nucleotide is added, the polymerase can remove it using 3′ → 5′ exonuclease activity, then resume synthesis.
Stepwise diagram of DNA polymerase proofreading during replication. It illustrates misincorporation, transfer to the exonuclease activity for removal of the mismatched nucleotide, and return to the polymerase active site to resume accurate extension. Source
Proofreading reduces the mutation rate by preventing mismatches from remaining in the newly synthesised DNA.
What happens to RNA primers (conceptual focus)
RNA primers are temporary. After DNA polymerase extends from a primer, the RNA segment must ultimately be replaced with DNA so the final product is an all-DNA strand. Different enzymes handle primer removal and replacement in prokaryotes versus eukaryotes, but the core idea is consistent: primers initiate, and DNA polymerase extends by adding to the 3′ end.
FAQ
Primers are usually short (often ~5–15 nucleotides in prokaryotes and ~10–30 in eukaryotes).
Length matters because primers must be long enough to bind stably and provide a reliable 3′-OH for extension, but short enough to be efficiently removed and replaced.
Primase (an RNA polymerase) can initiate nucleic acid synthesis without a pre-existing 3′ end.
DNA polymerase’s catalytic mechanism and active-site geometry require a paired primer end to correctly position the incoming dNTP for bond formation.
No. Many replicative polymerases proofread, but some specialised polymerases prioritise bypassing damage over accuracy.
Proofreading capability depends on whether the polymerase has 3′→5′ exonuclease activity.
Different organisms and cell compartments use different polymerases with distinct roles.
For example, bacteria commonly use DNA polymerase III for bulk synthesis, whereas eukaryotes rely heavily on polymerases such as $\delta$ and $\varepsilon$ during nuclear DNA replication.
Cells use protein–protein interactions to recruit DNA polymerase to the newly made primer.
Accessory factors (often sliding clamps and clamp loaders) can increase polymerase attachment and efficiency, helping rapid handoff from primase to the extending polymerase.
Practice Questions
Describe how DNA polymerase synthesises DNA from an RNA primer, including directionality and how accuracy is maintained. (5 marks)
DNA polymerase binds at the primer-template junction (1)
Adds dNTPs complementary to the template by base pairing (1)
Nucleotides are added to the 3′ end; synthesis is 5′→3′ (1)
Forms phosphodiester bonds to extend the strand (1)
Proofreading removes mispaired nucleotides via 3′→5′ exonuclease activity (1)
Explain why an RNA primer is required before DNA polymerase can synthesise a new DNA strand. (2 marks)
DNA polymerase cannot start synthesis de novo / requires an existing strand (1)
Primer provides a free 3′-OH for addition of DNA nucleotides / extension at 3′ end (1)
