Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2

In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2(+) causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (∼10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5′ single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase δ-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability

SDS–PAGE and western blot analysis of recombinant CeDna2 and CeRPA proteins

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic properties of the Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2"</p><p>Nucleic Acids Research 2005;33(4):1372-1383.</p><p>Published online 3 Mar 2005</p><p>PMCID:PMC552954.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Crude extracts, fractions obtained during each purification step, and purified wild-type and mutant CeDna2 enzymes were subjected to 8% SDS–PAGE. The gel was stained with Coomassie blue (left) and analyzed in western blot analysis (right) using monoclonal antibodies specific for the hexahitidine epitope. Lanes 1 and 8, crude extracts (50 μg for SDS–PAGE; 10 μg for western blot analysis) from uninfected insect cells; lanes 2 and 9, crude extracts (50 μg; 10 μg) from insect cells infected with baculovirus overproducing wild-type CeDna2; lanes 3 and 10, fractions (50 μg; 10 μg) obtained from Q-Sepharose column chromatography; lanes 4 and 11, active fractions (20 μg; 5 μg) from the Ni-NTA column; lanes 5 and 12 (1 μg; 200 ng), fractions from the anti-FLAG affinity column. Lanes 2–5 and 9–12, wild-type HF-CeDna2. Lanes 6 and 13, purified HF-CeDna2D320A (endonuclease-deficient; 1 μg and 200 ng, respectively); lanes 7 and 14, HF-CeDna2K678E (helicase-deficient; 1 μg and 200 ng, respectively). The numbers at the left of the Figure indicate the molecular size (in kDa) of marker proteins (indicated as M). The position of HF-CeDna2 is also indicated. () Purified recombinant RPA proteins (1 μg each) isolated from overproducing strains were subjected to 12% SDS–PAGE and the gel was stained with Coomassie blue. hRPA, human RPA; CeRPA, RPA; and ScRPA, RPA. The numbers at the left of the Figure indicate the molecular size (in kDa) of marker proteins (indicated as M)

Comparison of the cleavage of short 5′ free ssDNAs by CeDna2 and ScDna2

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic properties of the Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2"</p><p>Nucleic Acids Research 2005;33(4):1372-1383.</p><p>Published online 3 Mar 2005</p><p>PMCID:PMC552954.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The partial duplex substrates used were the same as depicted in . The two 5′ overhang substrates with 7 and 10 nt dT tails [(dT) and (dT), respectively] were used in this experiment and are as indicated. Reaction mixtures (20 μl) containing 15 fmol of the indicated substrates and 0.2–1 ng of HF-CeDna2 or ScDna2 were incubated at 37°C for 10 min in the presence of 5 mM MgCl. The cleavage products were then analyzed on a low-resolution 10% polyacrylamide gel