3.2 Replication Enzymes and Strand Direction

A DNA strand has a 5′ end associated with the fifth carbon of deoxyribose and a 3′ end with a free hydroxyl group on the third carbon. DNA polymerase catalyses addition to that 3′ hydroxyl, so a daughter strand extends only in the 5′→3′ direction. The enzyme therefore moves along its template in the opposite, 3′→5′ direction. Stating only that replication goes 5′→3′ is incomplete unless the answer identifies the newly synthesized strand.

The substrates are activated dNTPs. Each has a deoxyribose, a base and three phosphate groups. When a nucleotide is incorporated, cleavage of bonds associated with the outer phosphates helps drive formation of the phosphodiester bond. Polymerase cannot begin a strand from nothing: it needs an existing short nucleic-acid primer with a free 3′ end.

At one template, polymerase can follow helicase toward the moving fork while extending continuously. The daughter strand made this way is the leading strand. On the other antiparallel template, a polymerase cannot build toward the fork without violating the 5′→3′ rule. Synthesis instead restarts repeatedly near the fork and proceeds away from it, producing the lagging strand as short Okazaki fragments.

The names leading and lagging refer to newly synthesized strands, not permanent identities of the two parental strands across an entire chromosome. Replication usually proceeds bidirectionally from an origin. The same parental strand can serve as the leading-strand template at one fork and the lagging-strand template at the fork moving in the other direction. A correct diagram must therefore show both polarity and fork motion.

Single-strand binding proteins attach to exposed parental DNA, preventing the separated strands from re-pairing and reducing secondary structure that would obstruct enzymes. Topological strain also develops ahead of an unwinding fork; enzymes that relieve this strain are important in real replication systems. The IB mechanism focuses on helicase, primase, polymerases and ligase, but the fork should be understood as a coordinated protein assembly rather than a set of isolated enzymes.

Primase is an RNA polymerase that makes a short RNA primer complementary to the template. On the leading strand, one primer can support long continuous synthesis. On the lagging strand, each new Okazaki fragment requires another primer. Replicative DNA polymerase then adds dNTPs to each primer's 3′ end. The repeated need for primers is a consequence of strand geometry, not a different chemical rule for the lagging strand.

The RNA primers must not remain in a completed DNA molecule. Another DNA polymerase removes the RNA nucleotides and replaces them with DNA. This leaves adjacent DNA segments separated by nicks: the bases are present and paired, but the sugar–phosphate backbone is not covalently continuous. DNA ligase seals these nicks by catalysing phosphodiester bond formation. Ligase does not add a missing stretch of bases and does not join the two complementary strands to each other.

Proofreading depends on molecular fit. When a newly added base is mismatched, the geometry of the polymerase–DNA complex is disturbed. A proofreading activity can remove the incorrect nucleotide, after which synthesis resumes. Proofreading greatly lowers the error rate but cannot guarantee perfection. Any error that survives proofreading and repair becomes fixed as a mutation after another round of replication makes it part of both strands.