Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Abstract

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G 2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-␥ are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

“…7A, lanes 11-15 versus 16 -20). This observation is consistent with the previous findings that CAF-1 reassembles nucleosomes on daughter strands during DNA replication both in vitro and in vivo (15)(16)(17). These results suggest that the activity of FACT in nucleosome assembly on newly synthesized daughter strands is less significant than that of CAF-1.…”

“…Cell Cycle Analysis by Flow Cytometry-Flow cytometry was performed as previously described (17). For two-dimensional cell cycle analysis, cells were cultured in the presence of bromodeoxyuridine (BrdU; BD Biosciences) for 10 min, fixed in 70% ethanol, and stained with FITC-labeled anti-BrdU antibody (BD Biosciences) and propidium iodide.…”

“…CAF-1 is also required for the reassembly of nucleosomes into newly synthesized DNA duplexes in the cells (16,17). Based on functional and physical interactions between CAF-1 and proliferating cell nuclear antigen, a sliding clamp for eukaryotic DNA polymerases ␦ and ⑀, nucleosome reassembly is reported to be mechanistically coupled to DNA synthesis (18).…”

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

“…Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 assembles histones H3 and H4 onto newly synthesized DNA during replication and repair ( [1,2] and references in [3][4][5]). p150 utilizes its carboxylterminal third to bind p60 [6], and knocking down either subunit disrupts the chromatin assembly activity, delays cell cycle progression and leads to cell death [7][8][9].…”

Nucleosome assembly and epigenetic inheritance

“…Both RNAi mediated knockdown and overexpression of a dominant negative CAF-1 in cultured human cells result in defective assembly of newly replicated chromatin (Hoek and Stillman, 2003;Ye et al, 2003;Nabatiyan and Krude, 2004). Conditional depletion of CAF-1 p150 or p60 subunit in chicken DT40 cells leads to delayed S phase progression concomitant with slower DNA synthesis (Takami et al, 2007).…”