Distribution of DNA replication proteins in Drosophila cells

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 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.

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 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.

Figure 1

Drosophila and human PCNA are highly similar. (A)Protein sequence alignment of HsPCNA (human), DmPCNA (Drosophila) and ScPCNA (budding yeast). Residues identical to HsPCNA are boxed. Regions in HsPCNA that interact with the PBD of p21 [26] are in red. (B) A conserved PBD in proteins involved in DNA metabolism. Alignment of the PBD from various homologous proteins from different organisms. Accession numbers are indicated in the second column where available. Asterisk denotes the beginning or end of the protein. Identical residues are highlighted in red and conserved substitution in green. (Hs: Homo sapiens; Mm: Mus musculus; Xl: Xenopus laevis; Dm: Drosophila melanogaster; Ce: Caenorhabditis elegans; Sp: Schizosaccharomyces pombe; Sc: Saccharomyces cerevisiae; Mj: Methanococcus jannaschii).

Figure 2

Mammalian and Drosophila PCNA accumulate at RF interchangeably between these divergent organisms. Drosophila S2 and mammalian C2C12 cells were transfected, pulse labeled with BrdU to label sites of active DNA replication, and fixed with formaldehyde followed by immunostaining. (A) S2 cells expressing HsPCNA and coimmunostained with anti-PCNA (FL-261; Santa Cruz) and anti-BrdU antibodies. Anti-PCNA antibody (FL-261) specifically stains transfected cell (arrow) expressing HsPCNA. (B) S2 cells expressing HsPCNA (top) or GFP-HsPCNA (bottom) and immunostained with anti-BrdU. HsPCNA was detected as in (A). Overlay shows colocalization of HsPCNA and GFP-HsPCNA with BrdU foci. (C) C2C12 cells transfected with plasmid encoding GFP-DmPCNA and immunostained with anti-BrdU. Scale bar = 2 μm.

Figure 3

Subnuclear localization of PCNA-interacting proteins during S phase in Drosophila cells. (A) Plasmid constructs used to evaluate the association of mammalian replication proteins with RF in S2 cells. Mammalian cells expressing: NMT-GFP (B), GFP-HsLig (F). Drosophila cells expressing: NMT-GFP (C), Dnmt1 (D), GFP-HsLig (E), MTPBD-GFP (G-top panel) and HsLigPBD-GFP (G-bottom panel). RF were detected by BrdU incorporation and immunostaining with anti-BrdU. In (C) arrows in bottom panel show aggregation of NMT-GFP in large structures that are excluded from BrdU incorporation sites. In (D) top panel represents pattern observed in 80–90% of cells and bottom panel represents pattern observed in 10% of cells. Overlays show merge of green and red images. Scale bar = 2 μm.

Figure 4

Identification of the putative DNA Ligase I in Drosophila and its potential PBD. BLAST search of the D. melanogaster genome and EST database with DNA Ligase I (from different species) identified three similar sequences CG5602, CG17227 and CG12176. (A) Each of the putative DNA ligase protein sequences identified in Drosophila was aligned pairwise with HsDNA Lig I. The percentage identity is indicated at the right. (B) Table showing percentage identity obtained from pairwise alignment of the three putative DNA ligase homologues in Drosophila with each of HsDNA Ligase I, III and IV. (C) Phylogenetic tree from the comparison of HsDNA ligases and the Drosophila homologues created in Lasergene program. Scale represents amino acid substitution). (D) Structure of DmDNA Lig I gene and predicted protein. The gene structure is illustrated at the top; numbers indicate nucleotide positions; white boxes represent introns. A region in the 5th exon (marked gray) was found to be absent in the cDNA clone. This region was not detected as an intron using algorithms to detect introns. In the middle the LD41868 cDNA clone obtained from ResGen. Translational start is shown by arrow and the protein produced from this cDNA clone is illustrated at the bottom. (E) PBD-like sequences identified in DmDNA Lig I protein. Asterisk indicates the beginning or end of the protein sequence. Other features as in Figure 1B.

Figure 5

Drosophila DNA Ligase I is targeted to RF in mouse but not in Drosophila cells. (A) Organization of domains in DmDNA Lig I is shown at the top and below the various deletions of DmDNA Lig I fused to GFP. The predicted molecular weight of the fusion proteins is indicated on the left. The targeting to RF of each fusion in mammalian and Drosophila cells is summarized on the right. (B and C) Subcellular localization of the DmDNA Lig I (GdL, B) and DmLigPBD-GFP (C) with respect to BrdU labeled RF in S2 cells (upper panel) and C2C12 cells (bottom panel). Scale Bar = 2 μm.