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Distribution of DNA replication proteins in Drosophila cells

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Item Type:Article
Title:Distribution of DNA replication proteins in Drosophila cells
Creators Name:Easwaran, H.P. and Leonhardt, H. and Cardoso, M.C.
Abstract:BACKGROUND: DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. RESULTS: Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any association with RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to associate with RF in Drosophila and mammalian cells. Surprisingly, no association with RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I associated with RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. CONCLUSION: We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of associating with RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very transient interactions at the RF.
Keywords:Amino Acid Sequence, Cell Line, DNA (Cytosine-5-)-Methyltransferase, DNA Ligases, DNA Replication, Molecular Sequence Data, Proliferating Cell Nuclear Antigen, Saccharomyces Cerevisiae, Animals, Drosophila Melanogaster, Mice
Source:BMC Cell Biology
Publisher:BioMed Central
Page Range:42
Date:15 October 2007
Official Publication:https://doi.org/10.1186/1471-2121-8-42
PubMed:View item in PubMed

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