OKAZAKI FRAGMENT PROCESSING AND TRINUCLEOTIDE REPEAT STABILITY IN S. CEREVISIAE: A CRITICAL ROLE OF THE N-TERMINAL DOMAIN OF DNA2 IN MAINTENANCE OF TRINUCLEOTIDE REPEATS

초록

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 dissociates 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 primers from Okazaki fragments, is inefficient on primers consisting of TNRs. Here, we show that the enzymatic properties of Dna2 provide a built-in mechanism by which TNRs is stably maintained in vitro during Okazaki fragment metabolism in yeast. The Saccharomyces cerevisiae Dna2 helicase/endonuclease is essential for cell viability and is well suited to remove RNA primers of Okazaki fragments. The two endonucleases, 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. In addition to the endonuclease and helicase activities, which are encoded by the central and C-terminal domains, respectively, N-terminal 45-kDa domain of Dna2 was also essential for the optimal growth of cells. However, the mutant Dna2 protein lacking the N-terminal 45-kDa retained wild-like enzymatic activities. The analysis of biochemical activity of this mutant protein suggests that the N-terminal 45-kDa domain is critical for targeting the enzyme to the flap containing a secondary structure. The helicase activity resolves the secondary structure, facilitating the subsequent cleavage of the flap. This mechanism requires coordinated action of three activites of this enzyme, and is assisted by RPA. Our results demonstrate that the malfunction of Dna2 during processing of Okazaki fragments in eukaryot