Abstract
Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes.
Highlights
The significance of physical/genetic interactions between Dna2 and Fen1 is poorly understood
The mutual stimulation observed between Rad27 and Dna2 and the trans-autostimulation of Rad27 or human FEN1 leads to the efficient processing of Okazaki fragments in eukaryotes
We examined the genetic and physical interactions between Dna2 and Rad27 and showed that Rad27 stimulated the endonuclease activity of Dna2 and that Rad27 is a trans-autostimulatory enzyme using two catalytically defective Rad27 proteins
Summary
The significance of physical/genetic interactions between Dna and Fen is poorly understood. Dna and Rad (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. We addressed the functional consequences of these interactions by examining whether purified Rad of Saccharomyces cerevisiae affects the enzymatic activity of Dna and vice versa For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna, but failed to do so with Dna2⌬405N that lacks the N-terminal 405 amino acids. RPA, which stimulates the Dna2-catalyzed cleavage of long flaps [13], increased the Fen1catalyzed cleavage of short flaps that do not bind RPA [33] This indicates that interactions between the major proteins involved in Okazaki fragment processing are important for the efficient removal of flaps. The mutual stimulation observed between Rad and Dna and the trans-autostimulation of Rad or human FEN1 (hFEN1) leads to the efficient processing of Okazaki fragments in eukaryotes
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