title: Poly(ADP-ribose) polymerase at active centromeres and neocentromeres at metaphase.
abstract: A double-stranded 9 bp GTGAAAAAG pJ alpha sequence found in human centromeric alpha-satellite DNA and a 28 bp ATGTATATATGTGTATATAGACATAAAT tandemly repeated AT28 sequence found within a cloned neo- centromere DNA have each allowed the affinity purification of a nuclear protein that we have identified as poly(ADP-ribose) polymerase (PARP). Use of other related or unrelated oligonucleotide sequences as affinity substrates has indicated either significantly reduced or no detectable PARP purification, suggesting preferential but not absolute sequence-specific binding. Immunofluorescence analysis of human and sheep metaphase cells using a polyclonal anti-PARP antibody revealed centromeric localization of PARP, with diffuse signals also seen on the chromosome arms. Similar results were observed for mouse chromosomes except for a significantly enlarged PARP-binding region around the core centromere-active domain, suggesting possible 'spreading' of PARP into surrounding non-core centromeric domains. Enhanced PARP signals were also observed on alpha-satellite-negative human neo- centromeres and on the active but not the inactive alpha-satellite-containing centromere of a human dicentric chromosome. PARP signals were absent from the q12 heterochromatin of the Y chromosome, suggesting a correlation of PARP binding with centromere function that is independent of heterochromatic properties. Preliminary cell cycle analysis indicates detectable centromeric association of PARP during S/G(2)phase and that the total proportion of PARP that is centromeric is relatively low. Strong binding of PARP to different centromere sequence motifs may offer a versatile mechanism of mammalian centromere recognition that is independent of primary DNA sequences.
authors.affiliation: The Murdoch Institute, Royal Children's Hospital, Flemington Road, Parkville 3052, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Earle
authors.forename: A
authors.initials: A
authors.lastname: Saxena
authors.forename: A
authors.initials: A
authors.lastname: MacDonald
authors.forename: D F
authors.initials: DF
authors.lastname: Hudson
authors.forename: L G
authors.initials: LG
authors.lastname: Shaffer
authors.forename: R
authors.initials: R
authors.lastname: Saffery
authors.forename: M R
authors.initials: MR
authors.lastname: Cancilla
authors.forename: S M
authors.initials: SM
authors.lastname: Cutts
authors.forename: E
authors.initials: E
authors.lastname: Howman
authors.forename: K H
authors.initials: KH
authors.lastname: Choo
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002503:Centromere / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D006367:HeLa Cells; D006570:Heterochromatin / Q000201:enzymology; D006801:Humans; D008677:Metaphase / Q000235:genetics*; D051379:Mice; D008970:Molecular Weight; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep
chemical_list: D006570:Heterochromatin; D011065:Poly(ADP-ribose) Polymerases