The endonuclease activity of the yeast Dna2 enzyme is essential in vivo

Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activ ity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 fu nction. Systematic site-directed mutations of amino acid residues in Saccha romyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; ( ii) inviable only when expression was repressed; (iii) inviable. Biochemica l analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but w ere impaired in their endonuclease activity. Cells expressing mutant Dna2 e nzymes partially impaired in endonuclease activity were viable, but were un able to grow when expression of their mutant Dna2 enzymes was further reduc ed. Their growth was restored when the mutant Dna2 proteins decreased in nu clease activity were induced to overexpress, In contrast, mutant Dna2 prote ins lacking endonuclease activity did not allow cells to grow under any con ditions tested. These in vivo and in vitro results demonstrate that the end onuclease activity of Dna2 is essential for Okazaki fragment processing.