A pea homologue of human DNA helicase I is localized within the dense fibrillar component of the nucleolus and stimulated by phosphorylation with CK2 and cdc2 protein kinases.
DNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP- and Mg(2+)-dependent DNA and RNA unwinding activities, as well as ssDNA-dependent ATPase activity. The direction of DNA unwinding was 3' to 5' along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre-rRNA processing.
Tuteja
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International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 070, India. narendra@icgeb.res.in
Beven
A F
AF
Shaw
P J
PJ
Tuteja
R
R
D000906:Antibodies; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002466:Cell Nucleolus / Q000201:enzymology* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity
D000906:Antibodies; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D016203:CDC2 Protein Kinase; C027093:TraI protein, E coli; D004265:DNA Helicases