Distribution of DNA replication proteins in Drosophila cells

Hariharan P Easwaran,M Cristina Cardoso,Heinrich Leonhardt

doi:10.1186/1471-2121-8-42

Hariharan P Easwaran, M Cristina Cardoso + Show 1 more

Open Access

https://doi.org/10.1186/1471-2121-8-42

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Abstract

BackgroundDNA 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.ResultsFluorescent 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 accumulation at 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 accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at 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.ConclusionWe 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 accumulating at 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.

Highlights

DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF)
PCNA is highly conserved in S. cerevisiae, D. melanogaster and mammals The mechanistic basis for the association of replication factors with RF has been proposed to be mediated via interaction of PCNA binding domain (PBD) with PCNA
To understand whether the PBD-mediated association of proteins with RF is conserved in evolution, we first analyzed the conservation of PCNA protein sequence from divergent eucaryotes, especially the regions shown to interact with the PBD of p21Cip1/Waf1 [26]

Summary

Introduction

DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). BMC Cell Biology 2007, 8:42 http://www.biomedcentral.com/1471-2121/8/42 tuted of a subchromosomal domain of adjacent replicons from the same chromosomal region that replicate together in time and space and their associated protein machines (replisomes). The existence of various patterns of RF has been demonstrated in living mammalian cells by labeling the RF with fluorescent proteins fused to core replication factors, like DNA Ligase I or PCNA [5,6,7,8]. Such studies have shown that replication proteins continuously assemble at many subchromosomal domains to form RF, and continuously disassemble from sites that have completed replication

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