AP Syllabus focus:
‘DNA ligase joins Okazaki fragments on the lagging strand by forming covalent bonds, creating a continuous DNA molecule.’
DNA replication produces new DNA in pieces on one strand, creating a backbone with gaps that must be sealed. DNA ligase is the enzyme that permanently connects these pieces to maintain chromosome integrity.
What DNA ligase is and where it acts
DNA ligase: an enzyme that catalyses formation of a covalent phosphodiester bond to seal a break (“nick”) in the sugar-phosphate backbone of DNA.
Ligase’s key role is at sites where DNA is already correctly base-paired but the backbone is discontinuous. During replication, this is most prominent on the lagging strand, which is synthesised as short DNA segments.
Replication proceeds at a fork where the leading strand is synthesized continuously, while the lagging strand is built discontinuously as Okazaki fragments. The diagram highlights why fragments leave discontinuities (“nicks”) in the sugar–phosphate backbone and shows DNA ligase sealing those gaps to produce one continuous strand. Source
The substrate ligase “sees”
Ligase does not join random DNA ends during replication; it seals a nick between:
The figure outlines the ATP-dependent nick-sealing mechanism of DNA ligase, showing activation of the 5′-phosphate and final attack by the 3′-OH to form a phosphodiester bond. This directly links the structural requirement (aligned 3′-OH and 5′-phosphate) to the covalent chemistry that restores an unbroken sugar–phosphate backbone. Source
a 3′-OH group at the end of one DNA fragment
an adjacent 5′-phosphate at the start of the next fragment
These ends are aligned on a complementary template, so the sequence is already accurate; the missing step is backbone continuity.
Okazaki fragments and the need for ligation
Okazaki fragment: a short stretch of newly synthesised DNA made discontinuously on the lagging strand that must be joined to neighbouring fragments to form one continuous strand.
Because the lagging strand is made in multiple fragments, replication initially yields many correctly paired pieces separated by nicks.
This schematic summarizes how Okazaki fragments are processed and then sealed, emphasizing that ligase acts after the DNA is properly base-paired but still contains a backbone discontinuity. It helps connect the concept of “nicks” to the specific enzymatic step where DNA ligase converts adjacent fragments into a continuous lagging strand. Source
If those nicks persist:
the DNA backbone remains fragile and can break under cellular stress
the strand cannot function as a single, continuous chromosome segment
downstream processes that require intact DNA (e.g., chromosome packaging) are disrupted
Ligase resolves this by converting a “stitched” set of fragments into one continuous molecule, matching the syllabus emphasis on forming covalent bonds that create continuity.
The chemical outcome: covalent bonding in the backbone
The DNA backbone consists of repeating sugar-phosphate units linked by phosphodiester bonds. Ligase catalyses the final covalent linkage that connects adjacent nucleotides across a nick:
the bond formed is between the 3′ carbon of one sugar and the 5′ phosphate of the next nucleotide
the result is an unbroken sugar-phosphate backbone across former fragment boundaries
This step is conceptually distinct from nucleotide addition during synthesis: polymerases extend DNA, but ligase seals pre-existing, properly aligned ends.
Energy requirement (high-yield concept)
Creating covalent bonds requires energy input. Cells supply this via an activated ligase intermediate (energy source differs by organism), ensuring the sealing reaction proceeds efficiently and irreversibly under cellular conditions.
Why ligase is essential for replication fidelity and chromosome stability
Ligase directly supports accurate genome duplication by ensuring that discontinuous synthesis still produces a complete, stable DNA molecule:
Structural integrity: prevents accumulation of single-strand breaks that can convert into double-strand breaks during chromosome handling
Functional continuity: enables the lagging strand to become a true complementary strand, not a collection of fragments
Genome maintenance: reduces opportunities for loss of genetic information at unsealed nicks
What “continuous DNA molecule” means in practice
After ligation, the lagging strand is indistinguishable (in backbone continuity) from the leading strand: both are continuous polymers capable of being packaged, segregated, and used as templates in future replication cycles.
FAQ
In replication, it mainly seals single-strand nicks. Double-strand break joining is typically handled by dedicated repair pathways, not routine replication sealing.
Many eukaryotic ligases use ATP, while many bacterial ligases use NAD$^+$. Both provide energy to activate the phosphate for bond formation.
Not efficiently in cells unless ends are compatible and correctly aligned. Replication ligation is highly specific to adjacent nicks in aligned DNA on a template.
Unsealed nicks accumulate, increasing chromosome fragility and the chance of replication-associated breaks, which can trigger cell-cycle checkpoints or cell death.
In cloning, ligase can join compatible DNA ends (sticky or blunt) in vitro to create recombinant DNA, a broader use than sealing adjacent Okazaki fragments.
Practice Questions
State the role of DNA ligase during DNA replication and identify the strand where it is most needed. (2 marks)
Joins/seals Okazaki fragments by forming covalent (phosphodiester) bonds (1)
Acts mainly on the lagging strand (1)
Explain why DNA ligase is required after synthesis of Okazaki fragments and describe the specific chemical linkage it forms. (5 marks)
Lagging strand is synthesised discontinuously as Okazaki fragments (1)
After synthesis, fragments are base-paired but the sugar-phosphate backbone contains nicks (1)
Ligase seals nicks to make one continuous DNA molecule/strand (1)
Forms covalent phosphodiester bond between 3′-OH and 5′-phosphate ends of adjacent fragments (2)
