Immunofluorescence staining with the anti-hp150 monoclonal antibody (mAb1) confirmed that this distribution of hp150 overlapped with that of the GFP lineage marker (Physique?4B, bottom, GFP?+?hp150). two largest subunits of this complex concentrate at replication foci during S?phase (Krude, 1995; Martini et al., 1998; Shibahara and Stillman, 1999; Taddei et al., 1999) and at nucleotide excision repair (NER) sites outside of GSK-2881078 S?phase (Martini et al., 1998). Based on these properties of CAF-1 at the crossroads of various DNA metabolic pathways (Ridgway and Almouzni, 2000; Verreault, 2000), one would expect that a deficiency in its function would have a profound effect is not lethal and results in an increased sensitivity to UV irradiation and defects in transcriptional silencing in heterochromatic loci (Enomoto et al., 1997; Kaufman et al., 1997; Monson et al., 1997; Enomoto and Berman, 1998; Game and Kaufman, 1999). Based on these data, it is possible that in remains unclear. Remarkably, none of the chromatin assembly factors identified to date in has proved essential for nucleosome assembly or viability in this organism (Verreault, 2000). Key issues are thus raised concerning chromatin assembly factors and, more specifically, histone deposition factors and their exact function in different organisms and in various cell cycle contexts. p150 (xp150) CAF-1. Novel conserved dimerization properties of this subunit were discovered and their importance for CAF-1 function was assessed. A domain name of 36 amino acids not present in other known proteins to date, critical for p150 dimerization, was found. This permitted the design of a dominant-negative strategy to assess the specific role of p150 CAF-1 and under conditions ensuring maximum specificity. This study demonstrates a critical role for the largest subunit of CAF-1 during early embryonic development. Results Cloning and characterization of the Xenopus p150 CAF-1 homologue A yeast two-hybrid screen was carried out using as bait a portion (C-terminus) of the largest subunit of human CAF-1 (hp150 CAF-1) and, as prey, a oocyte cDNA library (Iouzalen et al., 1998). We did not retrieve the p60 homologue in this screen. This may be due to a weak conversation with hp150, a low representation of p60 cDNA or the presence of the restriction site used to construct the library within the xp60 cDNA. Unexpectedly, this screen enabled us to obtain the full-length sequence of a putative homologue of p150 CAF-1 in (Kaufman et al., 1995). In contrast, the N-terminal portion displayed weaker homology (Physique?1B). The sequence conservation in these domains suggested that our clone was the homologue of p150 CAF-1 and hence it was named xp150. Open in a separate window Open in a separate windows Fig. 1. A functional homologue of p150 CAF-1 in p150 (xp150) CAF-1 obtained using ClustalW and Boxshade programs (BCM and ISREC web sites). The amino acid identity is black boxed and similarity is usually shown by grey boxes. The position of the KER and ED boxes (Kaufman et al., 1995) is usually indicated on the side. (B)?Comparative schematic representation of the domain organization of human and p150. The percentage similarity/identity in the N- and C-terminal ends is usually indicated above the arrows delineating areas of comparison. Residues delimiting domains are indicated for each species. P, PEST domain name; KER, KER domain name; ED, ED domain name (Kaufman et al., 1995). (C)?Depletion GSK-2881078 of xp150 impairs chromatin assembly coupled to DNA repair. Top: western blot analysis of a egg extract (HSE) depleted of xp150. Anti-xp150 antibody (serum 566, 1/1000) and anti-PCNA antibody (PC10, DAKO) were used for detection. Lane?1, HSE depleted with control IgG; lane?2, HSE depleted with pre-immune serum; lane?3, HSE depleted with affinity-purified anti-xp150 antibody; lane?4, HSE depleted with anti-xp150 serum; lane?5, HSE diluted 1/10; lane?6, undiluted HSE equivalent to the depleted extract. Bottom: analysis of chromatin assembly by supercoiling on control and UV-irradiated DNA. The pBscript plasmid mock treated (C) or UV irradiated (+) (500?J/m2) (Gaillard et al., 1996) was incubated for 3?h GSK-2881078 at 23C in HSE, mock-depleted HSE or HSE depleted with anti-xp150 antibody. Alternatively, the DNA was incubated for 3?h at 37C in Slc3a2 S100 human cytosolic extract (Smith and Stillman, 1989) or S100 extract complemented with HSE treated as indicated. [-32P]dCTP was added to all samples to follow DNA repair synthesis. The migration of calm/nicked (Ir,II) and GSK-2881078 supercoiled DNA (I) is usually indicated. (D)?p150 complements S100 extracts for chromatin assembly. As in (A),.
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