Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity

Abstract

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.

FIGURE 1.

Mechanistic similarities and enzymatic differences between prokaryotic and eukaryotic lagging strand maturation. After replication initiation (represented by the red line) by DNA primases, pol III (prokaryotes) and pol δ (eukaryotes) take over replication elongation. The polymerases, along with their processivity clamps, β (prokaryotes) and PCNA (eukaryotes), are loaded onto the DNA with the help of the five-subunit clamp loaders, the clamp loader (prokaryotes) and replication factor C (RFC; eukaryotes). The single-strand binding proteins, SSB (prokaryotes) and RPA (eukaryotes), which help to protect the naked DNA template, are displaced as the replisome moves on the DNA. On encountering a downstream Okazaki fragment, pol III is disengaged, and pol I takes over the lagging strand maturation. pol I and pol δ, both having a 3′–5′-exonuclease for proofreading, can displace the 5′-end of the RNA primer on the downstream Okazaki fragment into a flap structure. This flap is cleaved by pol I 5′–3′-exonuclease activity (prokaryotes) and FEN1 5′–3′-endonuclease activity (eukaryotes) to create a nick that is subsequently sealed by LigI to complete the maturation process.

FIGURE 2.

Mechanism of the short and long flap pathways. The red line represents the initial RNA/DNA primer synthesized by pol α, and the black lines represent the nucleotides added by pol δ. RFC, replication factor C.

FIGURE 3.

The long flap pathway promotes genome stability by increasing the fidelity of the replication process. The dark blue lines on the DNA substrate represent the primer nucleotides added by pol α, and the red stars represent mismatches on the downstream Okazaki DNA sequence.