Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

Abstract

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells

“…This directly links pRb, DNA repair and UV radiationinduced apoptosis. pRb family members have been shown to co-localize with chromatin assembly factor-1 (CAF-1) (Kennedy et al, 2000), a protein important in NER of UV-induced lesions (Game and Kaufman, 1999;Martini et al, 1998). Indeed, in yeast, CAF-1 mutants are hypersensitive to UV radiation (Game and Kaufman, 1999).…”

Chromatin disassembly and reassembly during DNA repair

“…PCNA is required for the repair synthesis step of NER [53]. In turn, PCNA recruits CAF-1 to reassemble chromatin following NER [17,54,55].…”

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…Immunofluorescence-For visualizing the proliferating cell nuclear antigen (PCNA (53)) and Mcm2-7 on chromatin, cells were pre-extracted in 0.5% Triton X-100 in Cytoskeletal buffer (10 mM PIPES, pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 ) for 5 min on ice to remove soluble proteins, then fixed in 1.7% paraformaldehyde, and chilled, and methanol was extracted. Immunofluorescence was carried out using standard procedures (48).…”