title: Characterization of the endonuclease and ATP-dependent flap endo/exonuclease of Dna2.
abstract: Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5' single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5' single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.
authors.affiliation: Braun Laboratories, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Fortini
authors.forename: Subhash
authors.initials: S
authors.lastname: Pokharel
authors.forename: Piotr
authors.initials: P
authors.lastname: Polaczek
authors.forename: Lata
authors.initials: L
authors.lastname: Balakrishnan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Judith L
authors.initials: JL
authors.lastname: Campbell
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication / Q000502:physiology; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense
chemical_list: D004277:DNA, Single-Stranded; D008345:Manganese; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C509908:DNA2 protein, human