PHYSICAL AND FUNCTIONAL CROSSTALK BETWEEN NUCLEOTIDE EXCISION REPAIR AND DNA DAMAGE CHECKPOINT

Marco Muzi-Falconi,  Michele Giannattasio , Federico Lazzaro, Maria Pia Longhese ^§ and Paolo Plevani *.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy; ^§ Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy 
* paolo.plevani@unimi.it

INTRODUCTION. DNA damage checkpoints are surveillance mechanisms that ensure a proper cellular response to genotoxic insults. Malfunctioning of such systems is linked to premature aging, uncontrolled cellular proliferation, genome instability and, ultimately, tumor development (1). The cellular response to DNA damage is usually described as a signal transduction cascade, where sensor proteins detect a lesion in the double helix and elicit a signal, which is relayed to several effectors through the activity of different protein kinases. One of the aspects of the checkpoint cascade that is still poorly understood is the sensing step. How do cells realize that the DNA has been damaged, how are the lesions found within the genome and how is the first kinase in the cascade activated ? Checkpoint proteins are unlikely capable of recognizing directly all sorts of DNA lesions, which, even though they are of ample structural variety, trigger the same unique checkpoint response. An attractive possibility relies on DNA repair proteins attacking the lesions and providing common substrates to checkpoint factors. We have found a tight physical and functional connection between Nucleotide Excision Repair (NER) and checkpoint factors in response to UV damage. Our findings suggest a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndromes patients (2).

RESULTS AND DISCUSSION. Through a specific genetic screening, we isolated the cdu1-1 Saccharomyces cerevisiae mutant allele that is specifically defective in activating the checkpoint following UV lesions, but proficient in the response to alkylating agents and double strand breaks. CDU1 turned out to be identical to RAD14, the homologue of human XPA, involved in lesion recognition during NER, suggesting a similar role in checkpoint activation. Rad14 was also isolated as a partner of the Ddc1 checkpoint sensor protein (homologue to hRAD9) in a two hybrid screening, and physical interaction was proven by co-immmunoprecipitation and GST-pull down experiments. These last findings represent the first evidence of physical interactions between NER and checkpoint factors. To shed some light on the crosstalk between NER and checkpoint mechanisms, we tested at what step of the signal transduction cascade NER intervenes and we investigated what NER function is required for checkpoint activation. By measuring the phosphorylation state of critical checkpoint factors in several yeast rad strains, we found that NER functions are required to drive the earliest event in the signaling cascade, namely Ddc2 (homologue to hATRIP) phosphorylation. Null and point mutations (rad14∆, rad2∆, rad3K48R) affecting the core NER machinery abolish checkpoint responses to UV and their characterization suggest that lesion processing is needed for checkpoint activation. The possibility that NER factors may be involved in the actual recruitment of checkpoint proteins onto damaged DNA was examined by assessing DNA loading of Ddc1 and Ddc2. The UV damage-dependent association of these two checkpoint complexes to chromosomes was observed by chromatin co-sedimentation and chromosome spreading and is lost in rad14∆ and rad3K48R strains. These findings suggest that NER factors recruit checkpoint complexes in close proximity of the lesions and that subsequent processing is likely required to achieve stable DNA binding and kinase activation. While rad mutations affecting the whole NER response are defective in checkpoint activations, elimination of only the transcription coupled (rad26∆) or global genome (rad7∆) NER sub-pathways does not alter the checkpoint response. These last observations may offer an interpretation for the heterogeneity in cancer susceptibility observed in different NER syndromes patients.

REFERENCES.

1. Hartwell, L. (1992) Cell 71, 543-546.
2. Hoeijmakers, J. H. (2001) Nature 411, 366-374.