Okazaki fragment metabolism

Abstract

Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are ~1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

Figure 1.

Eukaryotic replisome. A DNA helicase initially unwinds the duplex DNA (red and blue strands) to separate the DNA and form a replication fork. The single-stranded DNA (ssDNA) is coated by the single-strand binding protein, replication protein A (RPA). On the leading strand, replication factor C (RFC) loads proliferating cell nuclear antigen (PCNA) and DNA polymerase ε to continuously synthesize the leading strand. The lagging strand is initially primed by DNA polymerase α (Pol α), which synthesizes a short RNA/DNA initiator primer (orange strand). RFC then displaces Pol α from the lagging strand to initiate the switch from the priming mode to the elongation mode. The initiator primer is extended by PCNA/DNA polymerase δ complex to form short segments of DNA known as Okazaki fragments.

Figure 2.

Okazaki fragment maturation. During Okazaki fragment maturation, (i) Pol δ displaces a short segment of the initiator primer into a 5′ flap; (ii) FEN1 recognizes the displaced flap, binds to the base of the flap and cleaves the flap; (iii) DNA ligase seals the nick; (iv) certain flaps are elongated by the action of the 5′–3′ helicase, Pif1; (v) the long flaps are stably coated by RPA; and (vi) Dna2 displaces RPA and cleaves the flap at multiple sites leaving a terminal product ∼5–6 nt in length.