title: Biochemical characterization of Warsaw breakage syndrome helicase.
abstract: Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5' single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3' single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2') substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2' substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.
authors.affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Wu
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Sommers
authors.forename: Irfan
authors.initials: I
authors.lastname: Khan
authors.forename: Johan P
authors.initials: JP
authors.lastname: de Winter
authors.forename: Robert M
authors.initials: RM
authors.lastname: Brosh
mesh_terms: D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049914:DNA Repair-Deficiency Disorders / Q000201:enzymology* / Q000235:genetics; D051856:Fanconi Anemia Complementation Group Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017384:Sequence Deletion; D013379:Substrate Specificity; D013577:Syndrome
chemical_list: C102108:BACH1 protein, human; D050976:Basic-Leucine Zipper Transcription Factors; D051856:Fanconi Anemia Complementation Group Proteins; D004247:DNA; D004265:DNA Helicases; C101830:DDX11 protein, human; D053487:DEAD-box RNA Helicases
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