The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

The transcriptional co-activator PGC-1␣ regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1␣ (NT-PGC-1␣) produced by alternative 3 splicing that introduces an in-frame stop codon into PGC-1␣ mRNA. The expressed protein includes the first 267 amino acids of PGC-1␣ and 3 additional amino acids from the splicing insert. NT-PGC-1␣ contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1␣ are dynamically regulated in the context of physiological signals that regulate fulllength PGC-1␣, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1␣ is a co-expressed, previously unrecognized form of PGC-1␣ with functions that are both unique from and complementary to PGC-1␣.

Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2) encodes the single P 5-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of SCARECROW (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by LOW PHOSPHATE ROOT 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stemcell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability.multicopper oxidase ͉ P5-type ATPase ͉ phosphate deficiency ͉ root development ͉ SCARECROW

Background: Plant-specific IQD genes encode putative CaM targets of unknown functions. Results: IQD1 interacts with KLCR1, binds to Arabidopsis CaM/CMLs, and localizes to microtubules. Conclusion: IQD1 may act as a scaffold protein recruiting cargo to kinesin motors for directional transport along microtubules. Significance: This work provides novel insight into IQD function and a framework to study plant kinesin regulation.

Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication. We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product. Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair. Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity. Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development. HEX1 gene equivalents were found in all vertebrates examined. The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.

2 /M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G 2 /M checkpoint response. We document that MNNG induces a robust, dose-dependent G 2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G 2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMRdependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases. INTRODUCTIONCells are continually exposed to numerous forces and toxins capable of damaging DNA. To ensure maintenance of genome stability, cells have evolved a complex set of mechanisms to appropriately respond to genotoxic damage. Such responses include genome surveillance and DNA repair, activation of cell cycle checkpoints, and apoptosis. Tumor initiation and progression are directly linked to genomic instability and often correlate with loss of gene(s) involved in genome damage response (Hartwell and Kastan, 1994;Loeb et al., 2003). Paradoxically, many cancer treatment regimens induce DNA damage and exert their therapeutic effects through activation of growth arrest or apoptotic responses. Thus, elucidating the mechanisms and molecules that govern DNA damage response is key to understanding the molecular basis of both tumor formation and the therapeutic effects of many anticancer drugs.The nitrosourea N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) is a well characterized monofunctional DNA alkylating agent. The cytotoxic and mutagenic potential of MNNG is chiefly attributable to its ability to alkylate (methylate) the O 6 -position of guanine, resulting in formation of O 6 -methylguanine (O 6 MeG) adducts (Goldmacher et al., 1986;Karran and Bignami, 1992). O 6 MeG forces O 6 MeG-T mispairing after DNA replication due to blocking of a hydrogen bonding position involved in complementary base pairing. This mutagenic lesion is primarily repaired via direct demethylation by the DNA repair protein methylguanine-DNA methyltransferase (MGMT) (Lindahl et al., 1982). Moreover, loss of MGMT activity renders cells extremely sensitive to MNNG and like alkylators (Kalamegham et al., 1988), underscoring the role that O 6 MeG lesions play in triggering response to this drug. O 6 MeG lesions are also recognized and repaired by the mismatch repair (MMR) system (Griffin et al., 1994;Duckett et al., 1996).In response to MNNG and other methylators, cells undergo a robust G...

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

p53 plays an important role in response to ionizing radiation by regulating cell cycle progression and triggering apoptosis. These activities are controlled, in part, by the phosphorylation of p53 by the protein kinase ATM. Recent evidence indicates that the monofunctional DNA alkylating agent N-methyl-N -nitro-Nnitrosoguanidine (MNNG) also triggers up-regulation and phosphorylation of p53; however, the mechanism(s) responsible for this are unknown. We observed that in MNNG-treated normal human fibroblasts, up-regulation and phosphorylation of p53 was sensitive to the ATM kinase inhibitor wortmannin. ATM-deficient fibroblasts exhibited a delay in p53 up-regulation indicating a role for ATM in triggering the MNNG-induced response. Likewise, a mismatch repair (MMR)-deficient colorectal tumor line failed to show rapid up-regulation of p53. However, unlike ATM-deficient cells, these MMR-deficient cells displayed rapid phosphorylation of the p53 residue serine 15 after MNNG. In vitro kinase assays indicate that ATM is rapidly activated in both normal and MMR-deficient cells in response to MNNG. Using a number of morphological and biochemical approaches, we failed to observe MNNG-induced apoptosis in normal human fibroblasts, suggesting that apoptosis-induced DNA strand breaks are not required for the activation of ATM in response to MNNG. Comet assays indicated that strand breaks accumulated, and p53 up-regulation/ phosphorylation occurred quite rapidly (within 30 min) after MNNG treatment, suggesting that DNA strand breaks that arise during the repair process activate ATM. These findings indicate that ATM activation is not limited to the ionizing radiation-induced response and potentially plays an important role in response to DNA alkylation.