Single Strand Annealing and ATP-independent Strand Exchange Activities of Yeast and Human DNA2

Abstract

The Dna2 protein is a multifunctional enzyme with 5′-3′ DNA helicase, DNA-dependent ATPase, 3′ exo/endonuclease, and 5′ exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented. The Dna2 protein is a multifunctional enzyme with 5′-3′ DNA helicase, DNA-dependent ATPase, 3′ exo/endonuclease, and 5′ exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented. Yeast Dna2 protein (yDna2) 3The abbreviations used are: yDna2, yeast Dna2 protein; MOI, multiplicity of infection; nt, nucleotide; HA, hemagglutinin. is a helicase/nuclease involved in the maintenance of genome stability (1Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: 634-650Crossref Scopus (0) Google Scholar). A human ortholog of yDna2, hDna2, has recently been characterized and was found to have biochemical features remarkably similar to its yeast counterpart (2Masuda-Sasa T. Imamura O. Campbell J.L. Nucleic Acids Res. 2006; 34: 1865-1875Crossref PubMed Scopus (85) Google Scholar, 3Kim J.H. Kim H.D. Ryu G.H. Kim D.H. Hurwitz J. Seo Y.S. Nucleic Acids Res. 2006; 34: 1854-1864Crossref PubMed Scopus (52) Google Scholar). When DNA2 is deleted from otherwise wild-type yeast cells, cells are inviable, suggesting that DNA2 is essential. However, the inviability of a dna2Δ strain can be suppressed by deletion of PIF1, encoding another helicase, and therefore, under this condition, DNA2 is not essential (4Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar). The helicase activity modulates the preference of Dna2 for substrates with specific structures (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Recessive mutations in the helicase domain are lethal, suggesting that the helicase is essential under most conditions (6Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In some circumstances, however, full helicase activity is not essential for viability (7Formosa T. Nitiss T. Genetics. 1999; 151: 1459-1470Crossref PubMed Google Scholar). Mutations eliminating nuclease activity are lethal but are deleterious even in the presence of wild-type Dna2 (8Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar, 9Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The precise biological role of Dna2 is not clear. The recently described comprehensive genomic integrity network in yeast, derived from a screen for genes synthetically lethal with dna2, points to not just one, but to a variety of cellular processes in which Dna2 may function, including DNA replication, DNA repair, and chromatin dynamics (1Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: 634-650Crossref Scopus (0) Google Scholar). The proposed role of Dna2 in Okazaki fragment processing has been studied most extensively (10Budd M.E. Campbell J.L. Mol. Cell Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). As a result of biochemical reconstitution studies one may conclude that Dna2 has an important role in the nucleolytic removal of RNA/DNA primers during the maturation of intermediates in lagging strand DNA synthesis (11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar, 12Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (174) Google Scholar). In this scenario, DNA polymerase δ (pol δ) is proposed to displace a 5′ RNA/DNA flap at the terminus of the previously synthesized Okazaki fragment (11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). The principal nuclease involved in the removal of the flap in preparation for joining of the fragments is FEN1 (11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar), the product of the RAD27 gene. Normally, pol δ and FEN1 activity are strictly coordinated to ensure efficient flap removal (12Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (174) Google Scholar). However, FEN1 is very inefficient in cleaving long flap structures bound tightly to RPA (11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Such flaps may arise during replication due to excessive strand displacement, which can result either from failure to properly coordinate FEN1 and pol δ, or to other disruptions of the polymerase cycle. Dna2, on the other hand, is well suited to perform the task of long flap cleavage. The helicase activity allows the processing of flaps containing duplex foldbacks that might arise when copying sequences containing repeats (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The nuclease activity requires long flaps and is stimulated by RPA (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). Therefore, the essential function of DNA2 may be the processing of long flap structures and/or structured flaps, providing an optimum substrate for FEN1, and thus preventing replication fork arrest (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (174) Google Scholar, 16Kao H.I. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 50840-50849Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). While consistent with biochemical reconstitution experiments, this model is also supported by genetic evidence. First, dna2 is synthetically lethal with rad27 (10Budd M.E. Campbell J.L. Mol. Cell Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). Overproduction of FEN1 leads to suppression of dna2-1 lethality. This may suggest that increase in gene dosage of RAD27 compensates for DNA2 deficiency, likely caused by either increased processing of the long flaps or to decreased strand displacement by pol δ (10Budd M.E. Campbell J.L. Mol. Cell Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). Second, rad27 pol3-01, and dna2-1 pol3-01 double mutants are both inviable (1Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: 634-650Crossref Scopus (0) Google Scholar, 15Jin Y.H. Ayyagari R. Resnick M.A. Gordenin D.A. Burgers P.M.J. J. Biol. Chem. 2003; 278: 1626-1633Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This may suggest that the absence of pol δ 3′ to 5′ exonuclease activity leads to excessive strand displacement and creation of long flap substrates that would require the cooperative action of FEN1 and Dna2 for processing (14Ayyagari R. Gomes X.V. Gordenin D.A. Burgers P.M.J. J. Biol. Chem. 2003; 278: 1618-1625Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Moreover, lethal interactions are observed, not just between DNA2 and RAD27, but between DNA2 and most of the key proteins participating in this process (1Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: 634-650Crossref Scopus (0) Google Scholar). Deletion of SGS1, which encodes a 3′-5′ helicase of the RecQ family (17Lu J. Mullen J.R. Brill S.J. Kleff S. Romero A.M. Sternglanz R. Nature. 1996; 383: PubMed Scopus Google Scholar), also involved in genome is synthetically lethal with mutations (1Budd M.E. Tong A.H. Polaczek P. Peng X. Boone C. Campbell J.L. PLoS Genet. 2005; 1: 634-650Crossref Scopus (0) Google Scholar, T. Campbell J.L. Res. 2003; PubMed Scopus Google Scholar). of the is that of dna2 is not to be involved in Okazaki fragment is to in repair of replication 2003; PubMed Scopus Google Scholar). However, double mutants are also inviable S. DNA 2005; PubMed Scopus Google Scholar). may functions in DNA in the absence of Dna2 other to for of dna2-1 the RecQ Overproduction of yeast was even in wild-type However, the human an ortholog of SGS1, the dna2-1 O. Campbell J.L. S. 2003; PubMed Scopus Google Scholar). We in suppression by the O. Campbell J.L. S. 2003; PubMed Scopus Google Scholar), another RecQ family but human dna2-1 S. J.A. Mol. Genet. 2004; PubMed Google Scholar). This the to activity of human or is for the and proteins to FEN1 S. J.A. Mol. Genet. 2004; PubMed Google Scholar, Bambara R.A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, one for the suppression of dna2-1 is that the increased of FEN1 may for the Dna2 well with the in in have that the helicase activity of may be important for the suppression of This is Dna2 and in 5′-3′ and is to two proteins with may the two activities to be with and and strand exchange activities on DNA C. 2005; PubMed Scopus Google Scholar, Mol. Biol. 2006; PubMed Scopus Google Scholar). The that activities might be involved in the of dna2-1 mutants by and to Dna2 has similar that both yeast and human Dna2 proteins are in single strand annealing and ATP-independent strand exchange on the of Dna2, and a model that the activities may to the efficient processing of flap structures in lagging strand replication This model may the that two of may perform leading to Dna2 and nuclease with at the terminus and at the and the proteins and the to described previously (2Masuda-Sasa T. Imamura O. Campbell J.L. Nucleic Acids Res. 2006; 34: 1865-1875Crossref PubMed Scopus (85) Google Scholar). at the terminus with one and at the terminus with a was in yeast and described previously for the wild-type Dna2 in and M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). was described previously M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). protein with at the terminus was in the This protein was of cells in containing in at with at an of and The was in of A and for at The was and bound to for at by with of A the in a and with of The was with of A and and a with the was with a from to The was at The in two and for and enzymes to to in containing and DNA synthesized by DNA used for strand strand and helicase and of was described previously (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). strand exchange and at a of to fork and in the to the that the of annealing activity of Dna2 was a containing the and of and ATP in at with and The with for at and a annealing activity of with DNA was the conditions The fragment used in was by O. Campbell J.L. S. 2003; PubMed Scopus Google a and primers used in 5′ of the fragment was (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). in was by and on of substrate was with in the the was by and by and described and DNA exchange activity of Dna2 was the conditions strand DNA fork substrate and in by and the DNA helicase activity of yDna2, strand was from the strand exchange on long DNA DNA was with exonuclease S. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). DNA was with and with in the by the with was at at a of of The of was on in the presence of and the by and DNA from this was used the duplex substrate in strand exchange by and 5′ of the fragment was exchange DNA and single-stranded DNA described by S. J. 2004; PubMed Scopus Google for with some DNA was with of single-stranded DNA in the strand exchange described with or and ATP in the was by of at by and the of DNA strand exchange and annealing in the absence of protein by or DNA with of single-stranded DNA at by DNA DNA activity of Dna2 was a The of DNA and of and ATP in for at with for at a of Yeast and annealing two and in otherwise the of and used to of the substrates As in both and the annealing of the two in the of the yeast the strand annealing is at of the protein and DNA and The yeast protein is efficient in strand annealing both in the absence and in the presence of of annealing by the human in is on the presence of both proteins are efficient in strand with to In the of yDna2, in the absence of to be a and at which strand annealing a increase with enzyme is with the We also the of on strand annealing by As in was the of ATP on the strand annealing activity of As in ATP is to strand annealing at and is to ATP strand annealing is also in the presence of ATP not inhibit the strand annealing activity of not We also the of a in the helicase domain A on strand This protein has or helicase activity but of nuclease activity M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). the yeast helicase is efficient in strand suggesting that ATP is not important for this activity of also that strand annealing can in the presence of an nuclease of is not strand annealing by not require which would be to be an essential in ATP and however, ATP inhibits strand annealing even in the the A in ATP and 9Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), can with In strand annealing is for but for The yeast protein is by and both yeast and human even in the A are by annealing not require either nuclease or helicase activities of by Yeast and the experiments, and are in strand exchange. and of under at a The a fork with 5′ and 3′ single-stranded adjacent to the duplex The is the substrate for (8Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar, M.E. Campbell J.L. S. 1995; PubMed Scopus Google Scholar). in the presence or absence of a with sequences to strand exchange result in the As in protein strand with the strand annealing the strand exchange both in the presence and the absence of We do a in strand exchange product in the presence of but the in product with to the nuclease activity of the protein in the presence of We suggest that the Dna2 nuclease on the long of the thus the of suggest that the product of strand exchange is and the is to the Dna2 nucleolytic cleavage. the of the activity is to be under conditions M.E. Campbell J.L. S. 1995; PubMed Scopus Google Scholar, S.H. E. K. J. S. Seo J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). As in conditions Dna2 helicase and of the is an increase in the product between and and by a in the product the helicase in the absence of a increase of the product during the of the In to is strand exchange product at the in the presence of ATP We that strand exchange is under conditions the helicase that in the presence of ATP and and that the at the of strand exchange is due to a two process of an by strand annealing of the and the with this that ATP inhibits the strand exchange As also strand exchange is efficient in the absence of and however, even in the absence of and thus in the absence of helicase is to strand exchange and was to the to is remarkably similar to in the strand exchange As in A and yDna2, strand exchange in the well in the presence of the strand exchange activity is that in the yeast however, to the conditions for strand exchange by the of the As in of the strand exchange We have in a under in the presence and the absence of of the substrate was to the strand exchange product and with yDna2, in the presence of nuclease activity the of strand exchange of the substrate and of to the at helicase activity of to inhibit strand the product with of the activity of and with helicase, the of ATP is to and likely to an of ATP on the strand exchange The of ATP and of that the strand exchange is a one an by of ATP on of Dna2 with inhibits the strand and strand exchange activities of This that ATP the of Dna2 with the DNA this the of to single-stranded to a fork and to a single-stranded duplex ATP inhibits the of Dna2 to of substrates both in the presence and in the absence of The on the of Dna2 with DNA may for the of ATP on the multiple activities of and with DNA Dna2 can strand annealing and strand exchange on used a duplex DNA fragment by for strand annealing and a for strand exchange Yeast Dna2 not annealing of the of the fragment under the conditions either when at or the strand exchange duplex DNA with single-stranded 5′ to Dna2 The and intermediates of strand exchange between this duplex and single-stranded DNA are in exchange in the would or on the to which exchange in annealing of the 5′ single-stranded to the single-stranded and of an in the in and and of a of the duplex with the in the of with the but the single-stranded DNA and by of DNA strand exchange to is the strand exchange that Dna2 to strand exchange either in the absence or presence of ATP at either to single-stranded or protein not annealing of the 5′ to We conclude from studies that Dna2, at under strand exchange not strand exchange of In this report two and activities of strand annealing and ATP-independent strand exchange on but not on DNA duplexes. from both yeast and human cells to have similar not require for strand the strand annealing of is of strand exchange by either however, are not by ATP inhibits both strand annealing and strand exchange for both enzymes This is in of the that ATP has been previously to inhibit the nuclease activity of Dna2, DNA studies suggest that ATP the of with single-stranded the of ATP on both at that ATP may to a in the enzyme that one or the other of its of annealing by ATP in the which be in the of that Dna2 may an ATP However, have not been to by to a the may not ATP The used here from used to both the and recently ATP-independent strand exchange activities involved in cellular and PubMed Scopus Google Scholar). in with but not of of intermediates in the PubMed Scopus Google Scholar). The used in are short to structures to Therefore, to strand annealing and strand exchange on These This not the that under proteins and/or other strand annealing and strand exchange activities be on the other hand, is that Dna2 to on which may during Okazaki fragment the by which Dna2 to flap processing during DNA replication and repair, of the biochemical activities of this multifunctional protein be including the strand annealing and strand exchange. is that the essential function of DNA2 in lagging strand DNA synthesis is to long flaps, which may arise due to polymerase by FEN1 FEN1, the nuclease in Okazaki fragment long flap substrates with in the presence of of FEN1 in or of the protein in the in this of Dna2, which in to FEN1 is stimulated by RPA (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). has thus been proposed that the nuclease activity of Dna2 a substrate by The Dna2 helicase may be for of structures that may arise in long flaps, in of and the nuclease might also be to the sequences that can to for FEN1 (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). While has been to that Dna2 FEN1 in to the coordination of interactions are the of that Dna2 and FEN1 coordination in some is not of the long flaps by Dna2 leads to a substrate and by FEN1 or Dna2 by FEN1 one of Dna2 do FEN1 but in other studies at of do the short flaps from Dna2 to substrates for FEN1 (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). the nuclease and helicase activities of Dna2 may not for of has the that the substrate for FEN1 in is not a single-stranded 5′ but a double flap with 3′ and 5′ flaps FEN1 a substrate a short nt, and and of the of the of the allows flaps are proposed to arise by of a by polymerase δ strand displacement activity by a 3′ and or While this may not at Okazaki is likely to arise is a pol and is not (12Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (174) Google Scholar, H.I. Bambara R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Y.H. R. Burgers P.M.J. Resnick M.A. Gordenin D.A. S. 2001; PubMed Scopus Google Scholar). yDna2, at protein FEN1 of flaps (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and was to of a in flap structures J.H. Kim H.D. Ryu G.H. Kim D.H. Hurwitz J. Seo Y.S. Nucleic Acids Res. 2006; 34: 1854-1864Crossref PubMed Scopus (52) Google Scholar). RPA on the of the J.H. Kim H.D. Ryu G.H. Kim D.H. Hurwitz J. Seo Y.S. Nucleic Acids Res. 2006; 34: 1854-1864Crossref PubMed Scopus (52) Google Scholar). Moreover, the nucleolytic of do not to to the FEN1 the intermediates not in to this is stimulated by Dna2 at Dna2 protein in (5Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This may be to the that this has been in the presence of of ATP and that the strand annealing activity is in the presence of at Dna2 can Dna2 strand annealing in the presence of ATP interactions have been in conditions both the helicase and nuclease activities of Dna2, and absence of ATP to the strand annealing be to the with ATP in the of FEN1 under conditions would an for the result that Dna2 was at protein in the presence of is to that ATP was found to be to of Dna2 to single-stranded This may suggest that ATP a in the the of the to of this The may also of the between Dna2 and the and have that helicase/nuclease and helicase, Dna2, also have annealing and strand exchange and also Dna2, are to with FEN1 and to FEN1 in The of FEN1 in was proposed that and of FEN1 may be due to increased of FEN1 at the and also to the of a dna2 suggesting that and Dna2 have either or the activities of Dna2 genes from and The and genes the Dna2 in either helicase or annealing and strand exchange activities are to gene is that the of the domain of with helicase or nuclease function, is to dna2, suggesting the helicase activity is not for The domain is for the FEN1 well strand exchange functions S. J.A. Mol. Genet. 2004; PubMed Google Scholar). In was that the helicase domain may be important for dna2 of a protein that helicase not the of a dna2-1 strain at O. Campbell J.L. S. 2003; PubMed Scopus Google Scholar). However, is that suppression was at well the of the dna2-1 by is that of full suppression of dna2 by was caused by a in another of the activities of due to of the stability of the or to the helicase Dna2 and biochemical may not require helicase activity in or nuclease and A of Dna2, and are strand exchange and in the of are for suppression of a Dna2 of dna2 not not not not in a can the of strand exchange activities described here to Okazaki fragment The role of Dna2 has been to removal of structures during processing of The activities may suggest the that can of structures that to in that the exchange activities of Dna2, and may the of flap 3′ and 5′ and/or 3′ and 5′ As a intermediates with the for FEN1 and/or pol δ would arise at leading to increased cleavage. with be by either Dna2 or the 3′ exonuclease of pol the by of FEN1 flap in to or in of a interactions and nuclease activities the of the increased of a strand annealing and strand exchange activities are most with processing of intermediates in Okazaki fragment The of the activities described here may for in of Dna2 to and/or of DNA structures that failure of Okazaki fragment of the replication fork in the presence of or replication may be important for the role of Dna2 (4Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar, Imamura O. Campbell J.L. Mol. Cell Biol. Scopus Google Scholar). We and of the for and of yeast and human