Cloning and Nucleotide Sequenceof the Genes Encoding Restriction-Modification System from Acidophilic Bacterium Acidocella facilis 22M

The gene encoding the Afa22MI restriction-modification system recognizing the sequence 5'-CGATCG-3' was cloned from Acidocella facilis 22M and sequenced. The cloned DNA fragment contained three genes encoding the Afa22MI methylase (M.Afa22MI) , the putative restriction endonuclease Afa22MI (R.Afa22MI) and a very short patch repair endonuclease (Afa22MI vsr) . M. Afa22MI gene has the conserved motifs of C5-cytosine methyltransferase. Afa22MI vsr gene was localized upstream of M. Afa22MI gene in opposite orientation, and an open reading frame of R. Afa22MI has about 53% sequence similarity to the amino acid sequence for the variable region of M.XorⅡ. Afa22MI vsr has about 66% sequence similarity to the amino acid sequence of XorII vsr which was associated M. XorII.CGATCGを認識する Afa22MI 制限修飾系遺伝子を好酸性細菌 Acidocella facilis 22M より、クローニングし、塩基配列を決定した。その結果、C5-シトシンメチラーゼに特徴的なモチーフが保存された。M.Afa22MI 遺伝子、その上流に、M.Afa22MI 遺伝子とは逆方向に、 very short patch repair endonuclease 様タンパク質遺伝子（Afa22MI vsr）、制限酵素（R.Afa22MI）遺伝子と推定されるオープンリーディングフレームが見出された。M.Afa22MI の推定アミノ酸配列は、Xanthomonas oryzae pv. oryzae 由来 M.XorII の配列と全体で約63%、veriable region 内で約53%の高い配列類似性を示した。また、Afa22MI usr の推定アミノ酸配列も、M.XorIIに付随する XorII vsr の配列と約66%の高い類似性を示した

ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors

Abstract Background Genomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness. Methods To elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy. Results Haploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin–proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion. Conclusions Our study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future