DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA

Robert (Rob) Bennett, G. T. Gabor, M. M. Merritt

    Research output: Contribution to journalArticle

    212 Citations (Scopus)

    Abstract

    Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononuclear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E- cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was a saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was ~10-9 M, and from 0.81 x 103 to 2.6 x 103 molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid-polydeoxythymidylic acid (poly[d(A)·d(T)], or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamine gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E- cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.

    Original languageEnglish (US)
    Pages (from-to)2182-2190
    Number of pages9
    JournalJournal of Clinical Investigation
    Volume76
    Issue number6
    StatePublished - 1985

    Fingerprint

    Leukocytes
    DNA
    Bacteriophage lambda
    Neutrophils
    Zonal Centrifugation
    Lymphocytes
    Ligands
    Collodion
    Cycloheximide
    Biotin
    Endocytosis
    Oligonucleotides
    Sodium Dodecyl Sulfate
    Electrophoresis
    Molecular Weight
    Gels
    Binding Sites
    Cell Membrane
    RNA
    Acids

    ASJC Scopus subject areas

    • Medicine(all)

    Cite this

    DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA. / Bennett, Robert (Rob); Gabor, G. T.; Merritt, M. M.

    In: Journal of Clinical Investigation, Vol. 76, No. 6, 1985, p. 2182-2190.

    Research output: Contribution to journalArticle

    Bennett, Robert (Rob) ; Gabor, G. T. ; Merritt, M. M. / DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA. In: Journal of Clinical Investigation. 1985 ; Vol. 76, No. 6. pp. 2182-2190.
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