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Edited by Wilhelm JustKeywords: ATM DNA damage and repair Ionizing radiation Chromosomal breakage and instability Cancer a b s t r a c tThe ability of our cells to maintain genomic integrity is fundamental for protection from cancer development. Central to this process is the ability of cells to recognize and repair DNA damage and progress through the cell cycle in a regulated and orderly manner. In addition, protection of chromosome ends through the proper assembly of telomeres prevents loss of genetic information and aberrant chromosome fusions. Cells derived from patients with ataxia-telangiectasia (A-T) show defects in cell cycle regulation, abnormal responses to DNA breakage, and chromosomal end-to-end fusions. The identification and characterization of the ATM (ataxia-telangiectasia, mutated) gene product has provided an essential tool for researchers in elucidating cellular mechanisms involved in cell cycle control, DNA repair, and chromosomal stability.

Recruitment of DNA repair factors and modulation of chromatin structure at sites of DNA double-strand breaks (DSBs) is a complex and highly orchestrated process. We developed a system that can induce DSBs rapidly at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Distinct advantages of this system over focus formation/disappearance assays for assessing DSB repair are demonstrated. Using ChIP, we found that nucleosomes are partially disassembled around DSBs during nonhomologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its arginine-glycine rich domain and is recruited to DSBs rapidly in an MRE11-NBS1-RAD50 complex-dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and the repair of the DSB, demonstrating the functional importance of nucleosome disruption in DSB repair and identifying a chromatin-remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/ H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for recruitment of replication protein A to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair.chromatin remodeling | DNA damage | I-PpoI | nucleosome disassembly

The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcriptionblocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)-and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.antibody microinjection ͉ DNA damage response ͉ RNA polymerase II ͉ nuclear export ͉ phosphorylation T he mechanisms by which DNA lesions are detected and how damage-response pathways are activated in cells are not well understood (1, 2). The stress-response kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) link DNA damage to p53 activation by phosphorylating the Ser-15 site of p53 (3-6). Although the ATM kinase is activated after exposure to ionizing radiation (7), the ATR kinase responds to agents that interfere with replication such as UV light and hydroxyurea (6). The tumor suppressor p53 is induced in response to a variety of different agents in all phases of the cell cycle (8,9). By acting as a transcription factor, p53 can induce the expression of gene products involved in DNA repair, cell cycle arrest, or apoptosis (10, 11). It can also regulate DNA repair and apoptosis by transcription-independent mechanisms (12). Under normal conditions, the p53 protein is rapidly targeted for nuclear export and degradation in a process regulated by the MDM2 protein (13). After cellular stress, the MDM2-mediated negative regulation of p53 is abrogated, and p53 proteins accumulate in the cell nucleus. This nuclear accumulation can be accomplished by (i) DNA damage-induced phosphorylation of p53 and MDM2, leading to the interference of the interaction between MDM2 and p53 (14); (ii) inactivation of proteasomes (15); or (iii) inhibition of the nuclear export machinery (13,16).DNA lesions that block RNA polymerase II induce the recruitment of transcription-coupled repair (TCR) and chromatin remodeling factors to recover RNA synthesis (17)(18)(19). Cells defective in TCR induce p53 and apoptosis at much lower doses than cells with proficient TCR, suggesting that lesions in the transcribed strand of active genes trigger these responses (9,(20)(21)(22). Although these studies have shown a correlation between blockage of ...

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