Dna2 of Saccharomyces cerevisiae Possesses a Single-stranded DNA-specific Endonuclease Activity That Is Able to Act on Double-stranded DNA in the Presence of ATP

Abstract

To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATPbinding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks.Maintaining the integrity of chromosomal DNA in eukaryotes is of critical importance to the cell and requires a series of complicated enzymatic processes. This is reflected in the complexity and redundancy of the enzyme systems that participate in DNA metabolism, such as replication, repair, and recombination (1, 2). In addition, DNA metabolism is tightly linked to cellular control pathways that regulate the cell division cycle (3-9). One of the enzymes required to achieve DNA replication, repair, or recombination is the DNA helicase, which uses the energy of ATP to translocate in a specific direction along a DNA strand melting the duplex regions it encounters (10 -14). The single-stranded DNA (ssDNA) 1 generated by the helicase is utilized by other enzymes that participate in the subsequent steps in DNA metabolic pathways. Recently, the DNA2 gene of Saccharomyces cerevisiae was implicated in chromosomal DNA replication (15, 16). DNA2 was originally identified by screening for cell division cycle mutants of S. cerevisiae and was shown to be essential for cell viability and to encode a 172-kDa protein with characteristic DNA helicase motifs (15). Analyses of a temperature-sensitive mutant of DNA2 demonstrated that the mutant cell arrested in the S phase of the cell cycle and was deficient in DNA synthesis but not RNA synthesis upon shift to the nonpermissive temperature (15). Immunoaffinity purified Dna2 fusion protein displayed a DNA-dependent ATPase activity as well as 3Ј to 5Ј DNA helicase activity specific for fork-structured substrates (15). In addition, a mutation in the ATP binding motif of DNA2 led to the inactivation of the ATPase and helicas...

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…The preparation of substrates and their labeling at either 5Ј-or 3Ј-end were as described previously (2,15). The structure and position of radioisotopic labels (indicated by asterisks) in substrates are described in each figure.…”

“…Substrates-All oligonucleotides used to construct DNA substrates were synthesized commercially (Genotech, Daejeon, Korea) and gel-purified prior to use as described previously (2). The preparation of substrates and their labeling at either 5Ј-or 3Ј-end were as described previously (2,15).…”

“…Recombinant wild type and mutant Dna2 proteins were prepared as described previously (2,7,13). Construction of the baculovirus that expressed HX-Dna2N (the N-terminal 405 amino acid fragment of Dna2) and purification of the recombinant protein were as described previously (34).…”

“…Dna2 is a highly conserved essential eukaryotic enzyme that contains ATP-dependent helicase and nuclease activities (1)(2)(3)(4)(5). Genetic and biochemical data indicate that its single-stranded (ss) 2 DNA-specific endonuclease activity is critical in both Okazaki fragment processing (5-7) and double strand break (DSB) repair (8 -11).…”

“…Genetic and biochemical data indicate that its single-stranded (ss) 2 DNA-specific endonuclease activity is critical in both Okazaki fragment processing (5-7) and double strand break (DSB) repair (8 -11). In Saccharomyces cerevisiae, the endonuclease activity of Dna2 is essential for viability (3,4), whereas its helicase activity is dispensable under some growth conditions (12,13).…”