Duplex DNA Is Unwound, and Daughter Strands Are Formed at the DNA Replication Fork

In order for duplex DNA to function as a template during replication, the two intertwined strands must be unwound, or melted, to make their bases available for pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands. This unwinding of the parent DNA strands is performed by enzymes called helicases. Unwinding begins at segments in a DNA molecule called replication origins, or simply origins. The nucleotide sequences of origins from different organisms vary greatly, although they usually contain AT-rich sequences. Once helicases have unwound the parent DNA at an origin, a specialized RNA polymerase called primase forms a short (~12-nucleotide) RNA primer complementary to the unwound template strands. The primer, still base-paired to its complementary DNA strand, is then elongated by DNA polymerase α for another 25 nucleotides or so, forming a primer made of RNA at the 5′ end and DNA at the 3′ end. This primer is further extended by DNA polymerase δ, thereby forming a new daughter strand.

The DNA region at which all these proteins come together to carry out the synthesis of daughter strands is called the replication fork. As replication proceeds, the replication fork and the associated proteins move away from the origin. As noted earlier, local unwinding of duplex DNA produces torsional stress, which is relieved by topoisomerase I. In order for DNA polymerases to move along and copy a duplex DNA, helicase must sequentially unwind the duplex and topoisomerase must remove the supercoils that form.

A major complication in the operation of a DNA replication fork arises from two properties of DNA: the two strands of the parent DNA duplex are antiparallel, and DNA polymerases (like RNA polymerases) can add nucleotides to the growing daughter strands only in the 5′→3′ direction. Synthesis of one daughter strand, called the leading strand, can proceed continuously from a single RNA primer in the 5′→3′ direction, the same direction as movement of the replication fork (Figure 5-29). The problem comes in synthesis of the other daughter strand, called the lagging strand.

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FIGURE 5-29 Leading-strand and lagging-strand DNA synthesis. Nucleotides are added by a DNA polymerase to each growing daughter strand in the 5′→3′ direction (indicated by arrowheads). The leading strand is synthesized continuously from a single RNA primer (red) at its 5′ end. The lagging strand is synthesized discontinuously from multiple RNA primers that are formed periodically as each new region of the parent duplex is unwound. Elongation of these primers initially produces Okazaki fragments. As each growing fragment approaches the previous primer, that primer is removed and the fragments are ligated. Repetition of this process eventually results in synthesis of the entire lagging strand.

Because growth of the lagging strand must occur in the 5′→3′ direction, copying of its template strand must somehow proceed in the opposite direction from the movement of the replication fork. A cell accomplishes this feat by synthesizing a new primer every 100 to 200 nucleotides on that template strand as more of the strand is exposed by unwinding. Each of these primers, base-paired to the template strand, is elongated in the 5′→3′ direction, forming discontinuous segments named Okazaki fragments after their discoverer, Reiji Okazaki (see Figure 5-29). The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighboring Okazaki fragment; finally, an enzyme called DNA ligase joins the adjacent fragments.