Okazaki Fragment Processing and Trinucleotide Instability in Eukaryotes

초록

Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNaseH1 and Fen-1 prior to the joining of Okazaki fragments. Despite the well-established in vitro roles of these two enzymes, genetic analysis in yeast revealed that null mutants of rither RNaseH1 or fen1, as well as RNaseH1/Fen1 double mutants, are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to remove RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonuclease Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by the single-stranded DNA binding protein, RPA. Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from and is more complicated than that ocuring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments. Instability of trinucleotide repeats (TNR) is responsible for at least 15 hereditary neurological disorders in humans. They vary in their sequences, are situated in a number of genes and affects gene expression in different ways. It is hypothesized that TNRs can expand or contract during lagging strand synthesis in two different modes; the newly synthesized Okazaki fragment dissiciates from template DNA, and forms a hairpin structure. The resumption of DNA synthesis leads to the repeats expansion. Alternatively, the flap endonuclease, which is responsible for removing RNA promers from Okazaki fragments, is inefficient on primers consisting of TNRs.