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The creation of accessible DNA in the context of chromatin is a key step in many DNA functions. To reveal how ATP-dependent chromatin remodeling activities impact DNA repair, we constructed mammalian genetic models for the INO80 chromatin remodeling complex and investigated the impact of loss of INO80 function on the repair of UV-induced photo lesions. We showed that deletion of two core components of the INO80 complex, INO80 and ARP5, significantly hampered cellular removal of UV-induced photo lesions but had no significant impact on the transcription of nucleotide excision repair (NER) factors. Loss of INO80 abolished the assembly of NER factors, suggesting that prior chromatin relaxation is important for the NER incision process. Ino80 and Arp5 are enriched to UV-damaged DNA in an NER-incision-independent fashion, suggesting that recruitment of the remodeling activity likely takes place during the initial stage of damage recognition. These results demonstrate a critical role of INO80 in creating DNA accessibility for the NER pathway and provide direct evidence that repair of UV lesions and perhaps most bulky adduct lesions requires chromatin reconfiguration.DNA damage | DNA repair | chromatin | remodeling T he highly condensed nature of the chromatin assembly restricts the interaction of DNA with most nuclear factors. To create accessible DNA for various nuclear events, chromatin dynamics is regulated by the coordinated actions of two types of cellular mechanisms, posttranslational modifications of the core histones and ATP-dependent chromatin remodeling. Whereas covalent histone modifications, instigated mainly from regulation of yeast gene transcription, have been extensively studied, less is known about the chromatin remodeling process and how chromatin remodeling impacts various nuclear events.ATP-dependent chromatin remodeling is catalyzed by a distinct class of enzymes that comprises four families of structurally related ATP-dependent protein complexes (1). Biological activities of these complexes, defined by a variety of in vitro assays, include disruption of histone-DNA contact within nucleosomes, increased accessibility of nucleosomal DNA to transcription factors or restriction endonucleases, and cis and trans movement of histone octamers (2, 3). The INO80 chromatin remodeling complex was identified from the ino80-1 mutant defective in inositol/choline response (4-6). It contains the Ino80 ATPase, which belongs to the SNF/SWI2 superfamily (7). The Ino80 ATPase associates with 14 proteins to form a 1-MDa complex exhibiting 3′-5′ helicase activity (6,8). The INO80 complex also contains three actinrelated proteins (ARPs), of which ARP5 and ARP8 are specific to the INO80 complex. Deletion of either INO80-specific ARP compromises the ATPase activity of the remaining complex and gives rise to DNA-damage-sensitive phenotypes indistinguishable to the INO80 null mutant (9). Purification of human INO80 revealed a complex with virtually identical core components and a role in transcription (10, 11), indicating ...

IntroductionChronic lymphocytic leukemia (CLL) is characterized by the gradual accumulation of abnormal neoplastic B cells in the bone marrow and blood. Although the early asymptomatic stage of CLL does not require treatment, the more aggressive forms of the disease cannot be cured by current treatment options. Current first-line treatment for many patients with CLL incorporates a fludarabine-based combination therapy. 1 However, disease relapse invariably occurs after treatment has been discontinued, and almost all patients with CLL will ultimately develop refractory disease. Therefore, new agents targeting the molecular mechanisms of CLL disease progression are highly desired.Antiapoptotic proteins of the B-cell lymphoma-2 (Bcl-2) family are overexpressed in most cases of CLL, and this overexpression is correlated with resistance to therapy and a poor prognosis. 2 Among the Bcl-2 family proteins, myeloid cell leukemia-1 (Mcl-1) has emerged as a significant antiapoptotic protein that promotes the survival of CLL cells both in vitro and in vivo. 3 Mcl-1 acts by preventing the proapoptotic proteins Bak and Bax from disrupting the mitochondrial membrane and initiating apoptosis. 4 Approaches that reduce Mcl-1 levels in CLL cells by direct methods such as small interfering RNA (siRNA) 5 or through indirect approaches to inhibit Mcl-1 transcription resulted in cell death. 6,7 Because the inhibition of apoptosis by Bcl-2 family proteins has been recognized as a distinct oncogenic function, 8,9 agents that antagonize the actions or diminish the expression of antiapoptotic proteins have been developed to induce apoptosis in CLL cells. These compounds, including oblimersen, an antisense oligonucleotide targeting Bcl-2 mRNA, 10 or the BH3 mimetics that interfere with the interaction of the proapoptotic and antiapoptotic proteins of the Bcl-2 family 11,12 are currently in clinical trials for treating CLL. A third strategy takes advantage of the fact that the key antiapoptotic protein in CLL, Mcl-1, is intrinsically unstable. 13 Transient exposure to flavopiridol, roscovitine, or SNS-032, small molecules that block transcription by inhibiting Cdk9, diminishes Mcl-1 transcripts and protein, with the subsequent induction of apoptosis. 6,7,14 These compounds are currently in clinical trials for treating CLL and other B-cell malignancies, and transient exposure schedules have generated responses in fludarabine-resistant disease. 15,16 Because Mcl-1 is thought to function as an oncogene on which CLL cells depend for survival, the striking activities generated by transient exposure to these transcription inhibitors may be attributed to the diminished Mcl-1 levels. This encouraged us to explore inhibition of translation, the subsequent step in protein expression, as an additional approach to activate cell death processes. 17 Earlier studies of inhibitors of translation showed that cycloheximide (CHX) was cytotoxic to CLL cells in vitro 18 and that puromycin enhanced the cytotoxic activity of fludarabine in CLL cells. 19 Recently,...

SUMMARY The DNA remodeling enzyme FANCM and its DNA-binding partner, FAAP24, constitute a complex involved in the activation of Fanconi Anemia (FA) DNA damage response mechanism, but neither gene has distinct patient mutants. In this study, we created isogenic models for both FANCM and FAAP24 and investigated their integrated functions in DNA damage response. We found that FANCM and FAAP24 coordinately facilitate FA pathway activation and suppress sister chromatid exchange. Importantly, we show that FANCM and FAAP24 possess non-overlapping functions such that FAAP24 promotes ATR-mediated checkpoint activation particularly in response to DNA crosslinking agents, whereas FANCM participates in recombination-independent interstrand crosslink repair by facilitating recruitment of lesion incision activities which requires its translocase activity. Our data suggest that FANCM and FAAP24 play multiple while not fully epistatic roles in maintaining genomic integrity.

Abstract2V -C -cyano-2V -deoxy-1-B-D-arabino -pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by B-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G 2 . The purpose of this investigation was to define the molecular basis for G 2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G 2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G 2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G 1 . Cell death was first evident in hematologic cell lines after G 1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G 2 . Some breaks were manifested as chromosomal aberrations when the G 2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies. (Cancer Res 2005; 65(15): 6874-81)