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
). 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
) 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 3′-5′ exonuclease (Saccharomyces cerevisiae
]) and a replication factor C (RF-C)-like protein (Rad24sc
). Protein kinases such as Mec1sc
appear to transduce signals from DNA damage sensors to the cell cycle machinery. Significant progress has been made in delineating the protein-protein interactions and phosphorylation events occurring among some of these factors and their potential interfaces with DNA repair (96
). However, the molecular nature of the links between the repair of specific DNA lesions and the DNA damage checkpoint machinery is not yet fully understood.
In addition to interconnections between DNA damage processing and the cell cycle checkpoint machinery, the way in which chromatin organization may influence both aspects is becoming of increasing interest (98
). The entire genome is packaged into chromatin (90
). This structure allows the compaction of DNA from the basic nucleosome unit (44
) up to a higher-order organization providing a potential range of reactivity (11
). Mutations affecting all acetylation sites in the N-terminal tail of yeast histone H4 give rise to a delay in the G2
and M phases of the cell cycle as a result of activation of the Rad9sc
-dependent DNA damage checkpoint (26
), suggesting that DNA integrity or cell cycle progression could be monitored by a marking at the chromatin level. In addition, a mechanistic link has been observed between DNA repair and chromatin assembly. Incubation of DNA damaged by UV irradiation in repair-competent cell-free extracts revealed that de novo nucleosome assembly occurs concomitantly with nucleotide excision repair (NER) (17
). A general model has been proposed for NER of DNA lesions within chromatin, in which the unfolding of nucleosomal structures facilitates access of repair enzymes to DNA and is followed by a rapid refolding (reviewed in references 15
, and 78
). The resetting of a preexisting chromatin structure during NER could relate to the mechanistic link between NER and chromatin assembly. An alternative function of de novo chromatin assembly may be to participate in the sensing of DNA damage.
The chromatin assembly pathway associated with NER is dependent on chromatin assembly factor 1 (CAF-1) (19
). This three-subunit complex functions as a histone chaperone, interacting with specific forms of histone H4 and H3 (91
). It is required for chromatin assembly during simian virus 40 DNA replication in vitro (35
), possibly relating to a general enrichment of this factor at replication foci in S-phase cells (39
). Remarkably, CAF-1 can also be recruited to chromatin during the repair of UV photoproducts outside of S phase (49
). This is consistent with the preservation of its capability to facilitate nucleosome formation when isolated from G1
- or G2
-phase nuclei (47
). In addition, genetic studies of the budding yeast revealed that although none of the genes corresponding to the individual CAF-1 subunits (CAC1
, and CAC3
) were essential, CAC
mutants were moderately sensitive to UV irradiation and exhibited gene silencing defects (13
). Together these data argue for a dual role for CAF-1 during both DNA replication and NER.
These data prompted us to explore the link(s) between CAF-1 and DNA damage processing at the biochemical level to identify partners and DNA structures critical for its recruitment. The involvement of CAF-1 in chromatin assembly during both DNA replication and NER suggested that this factor may sense, either directly or indirectly, the presence of a common nucleoprotein intermediate. This could be generated either through dual endonucleolytic cleavages in the damaged DNA strand during NER or at the 3′-hydroxyl termini of DNA replication forks. To test this hypothesis biochemically, we have created DNA substrates containing DNA damage in the form of single-strand breaks and gaps and show that they efficiently trigger the assembly of nucleosomal arrays. This nucleosome assembly pathway is dependent on CAF-1. Using the largest (p150) subunit of the CAF-1 complex as bait in a yeast two-hybrid screen, we have identified a specific interaction with PCNA. We further show that the N terminus of p150 interacts directly with specific sites on the outer front side of PCNA. To analyze the functional significance of this interaction in the context of DNA damage, we have developed an assay for factors recruited during DNA damage processing. Recruitment of PCNA and CAF-1 to damaged DNA is dependent on the number of DNA lesions and requires ATP. Furthermore, depletion of PCNA from a cell-free system disrupts chromatin assembly linked to single-strand break repair, and this defect can be rescued by complementation with recombinant PCNA. Thus PCNA, through CAF-1, can link multiple DNA repair pathways to chromatin assembly. The sliding clamp function of PCNA could account for the bidirectional propagation of nucleosomal arrays away from lesion sites during DNA repair. Our data suggest a possible mechanism for signaling the presence of DNA lesions to the DNA damage checkpoint machinery.