Okazaki fragment maturation: nucleases take centre stage

Abstract

Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells. Even in yeast, the Okazaki fragment maturation happens approximately a million times during a single round of DNA replication. Therefore, efficient processing of Okazaki fragments is vital for DNA replication and cell proliferation. During this process, primase-synthesized RNA/DNA primers are removed, and Okazaki fragments are joined into an intact lagging strand DNA. The processing of RNA/DNA primers requires a group of structure-specific nucleases typified by flap endonuclease 1 (FEN1). Here, we summarize the distinct roles of these nucleases in different pathways for removal of RNA/DNA primers. Recent findings reveal that Okazaki fragment maturation is highly coordinated. The dynamic interactions of polymerase δ, FEN1 and DNA ligase I with proliferating cell nuclear antigen allow these enzymes to act sequentially during Okazaki fragment maturation. Such protein-protein interactions may be regulated by post-translational modifications. We also discuss studies using mutant mouse models that suggest two distinct cancer etiological mechanisms arising from defects in different steps of Okazaki fragment maturation. Mutations that affect the efficiency of RNA primer removal may result in accumulation of unligated nicks and DNA double-strand breaks. These DNA strand breaks can cause varying forms of chromosome aberrations, contributing to development of cancer that associates with aneuploidy and gross chromosomal rearrangement. On the other hand, mutations that impair editing out of polymerase α incorporation errors result in cancer displaying a strong mutator phenotype.

Figure 1

Enzymes and reactions in the DNA replication fork. Major proteins factors present in a typical replication fork include: (i) mini-chromosome maintenance (MCM) proteins (six homo-subunits), which are helicases for opening up the DNA duplex to initiate a DNA replication fork; (ii) RPA, a single-stranded DNA binding protein to protect the DNA template from nuclease cleavage; (iii) primase (a complex of RNA polymerase and Pol α), which synthesizes RNA primers and a short DNA fragment to initiate Okazaki fragments; (iv) Pol δ, the DNA polymerase responsible for synthesizing the major portion of Okazaki fragments; (v) Pol ɛ, the DNA polymerase responsible for leading strand DNA synthesis; (vi) PCNA, which is the DNA clamp for the processivity of DNA polymerase and coordination of Okazaki fragment maturation processes; (vii) RFC, which is the clamp loader for PCNA to load onto DNA duplex; (viii) nucleases, including RNase H, DNA2 and FEN1 for removal of RNA primers and (ix) DNA Lig I, which joins processed Okazaki fragments into an intact DNA lagging strand. Black lines represent the DNA template, while pink ones are the newly synthesized DNA and light pink ones are the RNA primers.

Figure 2

Distinct roles of nucleases in sequential processing of RNA/DNA primers (α-segment). Upper panels show three different pathways involved in processing RNA primers: (i) FEN1-mediated short flap cleavage (middle); (ii) long flap degradation by sequential actions of DNA2 and FEN1 (right) and (iii) RNA primer removal by RNase H/FEN1 exonuclease (left). The bottom panel indicates the action of FEN1 or exonuclease to edit out incorporation errors of Pol α. Yellow circles represent ribonucleotides and cyan squares represent mismatched deoxyribonucleotides. Black lines correspond to DNA templates and pink lines correspond to newly synthesized DNA. Blue arrows indicate cleavage by RNase H or the exonuclease activity of FEN1 or exo-1.

Figure 3

Model for post-translational modifications that mediate the interaction between FEN1 and PCNA, and thus regulate the dynamic actions of FEN1 in processing of Okazaki fragment maturation. The Pol δ/PCNA complex drives the gap filling and formation of the flap structure in Okazaki fragment mutation. Methylated FEN1 is recruited to the replication fork by interacting with PCNA, replacing Pol δ. Methylation of FEN1 ensures its interaction with and stimulation by PCNA to remove the flap structure. After DNA flap cleavage, FEN1 undergoes de-methylation and subsequent phosphorylation, leading to FEN1 dissociation from the DNA nick. DNA Lig I is then recruited by interaction with PCNA and seals the nicks between the two Okazaki fragments.