A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

Abstract

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA-CAF-1 interaction in the context of DNA damage processing and checkpoint control.Sensing and signaling the presence of DNA damage to the cell cycle checkpoint machinery is crucial for the maintenance of genomic integrity and the regulation of cell cycle progression (12,25,61,97). Checkpoints respond to DNA damage by halting cell cycle progression, providing time for DNA repair. This strategy avoids the replication and segregation of damaged chromosomes which could otherwise lead to genomic instability. DNA damage is caused by physical and chemical agents as well as normal cellular processes including DNA replication and oxidative stress. A variety of distinct DNA repair mechanisms involving lesion-specific DNA damage recognition proteins have been characterized in eukaryotic cells (reviewed in reference 15). The DNA damage checkpoint machinery may recognize structural perturbations in DNA and/or components of the DNA damage processing machinery during specific phases of the cell cycle. Yeast model systems have proven powerful in identifying components of mitotic DNA damage checkpoint pathways (5,37,43,71,97) which, by analogy with signal transduction pathways, consist of sensor, transducer, and effector molecules. Several checkpoint proteins have been proposed to be directly involved in DNA damage recognition based on their similarity to proteins involved in DNA metabolism, including a structural relative of a 3Ј-5Ј exonuclease (Saccharomyces cerevisiae Rad17 [Rad17 sc ]) and a replication factor C (RF-C)-like protein (Rad24 sc ). Protein kinases such as Mec1 sc and Rad53 sc appear to transduce signals from DNA damage sensors to the cell cycle machinery. Significant progress has been made in delineating the proteinprotein interactions and phosphorylation events occurring among some of these factors and their potential interfaces with DNA repair (96). However, the mol...

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

“…Figure 7 shows that PCNA colocalizes partially with both WRN and CAF-1 following HU treatment. This is in agreement with previous reports that PCNA associates with CAF-1 in DNA replication (Moggs et al, 2000), after UV exposure (Green and Almouzni, 2003) process of double-strand breaks repair (Nabatiyan et al, 2006), and that PCNA interacts with WRN during replication (Rodriquez-Lopez et al, 2003). However, how WRN and PCNA coordinately interact with CAF-1 remains to be elucidated.…”

“…NER repairs singlestranded DNA lesions such as those incurred by exposure to ultraviolet light (UV) (Prakash and Prakash, 2000), and CAF-1 is capable of assembling chromatin coupled with NER in vitro (Gaillard et al, 1996). CAF-1 has also been localized to DNA templates undergoing NER, in a manner dependent upon proliferating-cell nuclear antigen (PCNA) (Moggs et al, 2000;Green and Almouzni, 2003). Accordingly, yeast deleted for CAF-1 components are hypersensitive to UV irradiation, indicating the importance of CAF-1 in surviving UV-induced DNA damage (Kaufman et al, 1997).…”

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].…”

Nucleosome assembly and epigenetic inheritance

“…Smith et al followed this RC nucleosome assembly activity in nuclear extract and isolated a three subunit protein complex termed as chromatin assembly factor I, CAF-I (Smith and Stillman, 1989). In cell free system, the loading of PCNA (proliferating cell nuclear antigen) is required for CAF-I mediated chromatin assembly (Shibahara and Stillman, 1999); furthermore, p150, the largest subunit of CAF-I complex directly interacts with PCNA (Shibahara and Stillman, 1999;Moggs et al, 2000). The interaction of p150 with histone H3/H4 through its KER and ED domains demonstrates CAF-1's histone chaperone activity (Kaufman et al, 1995).…”

“…PCNA directly interacts with a series of chromatin modifying enzymes, and potentially recruits them to replication foci. These factors include CAF-1 (Shibahara and Stillman 1999;Moggs et al, 2000), HDACs (Milutinovic et al, 2002), ATP dependent chromatin remodeling complex WSTF-SNF2h (Poot et al, 2004), histone lysine methyltransferse PR-SET7 (Jørgensen et al, 2007;Huen et al, 2008) and DNA methyltransferase DNMT1 (Leonhardt et al, 1992;Chuang et al, 1997). CAF-1 is also reported to interact with chromatin factors, including MBD1 (methyl CpG-binding protein 1) and SETDB1 during heterochromatin DNA replication (Reese et al, 2003;Sarraf and Stancheva, 2004).…”