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Analysis of AtZDP substrate specificity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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(English)Article in journal (Other academic) Published
URN: urn:nbn:se:uu:diva-97330OAI: oai:DiVA.org:uu-97330DiVA: diva2:172212
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2011-06-28Bibliographically approved
In thesis
1. AtZDP, a Plant 3' DNA Phosphatase, Involved in DNA Repair
Open this publication in new window or tab >>AtZDP, a Plant 3' DNA Phosphatase, Involved in DNA Repair
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA bases can be modified by endogenous agents (e.g. oxidized by products of respiration and photosynthesis or methylated by gene silencing processes) as well as by environmental agents (e.g. oxidized by UV light). In the process of removing modified bases, a 3’-phosphate group is sometimes left in the resulting gap, and has to be removed since it blocks the incorporation of a new nucleotide by DNA polymerase. The aim of this thesis was the characterization of AtZDP, a plant enzyme with a DNA 3’-phosphatase activity.

By homologous modeling, the existence of four domains was predicted in AtZDP, three independent zinc-finger and one DNA 3’-phosphatase domains. AtZDP was found to be localized in the nucleus by bimolecular fluorescence complementation. Western blotting analysis showed that the enzyme was ubiquitously expressed in plant tissues.

AtZDP was found in a 600,000 molecular-weight protein complex by gel chromatography and glycerol gradient sedimentation centrifugation. The fractions containing AtZDP in the complex displayed 3’-DNA phosphatase activity as shown by desphosphorylation of a DNA oligonucleotide with a 3’-phosphate terminus. Also fractions of the gel chromatography corresponding to lower molecular weight showed 3’-DNA phosphatase activity, but antibodies against AtZDP did not recognize this fraction inferring that in plants, at least another protein with similar activity exists.

In mammals, polynucleotide kinase, an enzyme with the same activity phosphatase activity as AtZDP, is involved in single-strand and double-strand repair pathways. To elucidate if AtZDP could be part of similar pathways, different double strand and single-strand oligonucleotides with 3’-phosphate termini were separately incubated with AtZDP. All substrates were dephosphorylated by AtZDP, assuming that this enzyme could potentially be involved in double-strand DNA repair.

A double-strand oligonucleotide containing a one-bp gap with a 3’-phosphate terminus was repaired by a leaf protein extract. The activities of a 3’-DNA phosphatase, a flap 5’ to 3’ endonuclease-like, a DNA polymerase and a DNA ligase were observed. The presence of these enzymes revealed that these damages are in plants predominantly repaired by long-patch base excision repair.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 39 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 362
Molecular biology, AtZDP, plant long patch Base Excision Repair, DNA 3' phosphate blocking lesions, DNA repair, Molekylärbiologi
urn:nbn:se:uu:diva-8907 (URN)978-91-554-7227-6 (ISBN)
Public defence
2008-06-04, C10:301, BMC, Uppsala, 13:15
Available from: 2008-05-14 Created: 2008-05-14Bibliographically approved

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Jemth, PerEngström, Åke
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