Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes

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pks+ Escherichia coli more prevalent in benign than malignant colorectal tumors

Molecular Biology Reports - Some E. coli strains that synthesize the toxin colibactin within the 54-kb pks island are being implicated in colorectal cancer (CRC) development. Here, the prevalence...     

Cleavage of a RNA analog containing uridine by a bifunctional dinuclear Zn(II) catalyst

The macrocyclic ligand, 1,4-bis((1-oxa-4,7,10-triazacyclododecan-7-yl)methyl)benzene (L1) is prepared. L1 binds two Zn(II) ions at neutral pH to form Zn₂(L1) as studied by using pH-potentiometric titrations. Zn₂(L1) binds two uridines at pH 7.0, I = 0.100 M (NaCl) and the mononuclear analog Zn(L2) (L2 = 1-oxa-4,7,10-triazacyclododecane) binds a single uridine; dissociation constants for both complexes are in the millimolar range. Both complexes promote the cleavage of a simple RNA analog lacking a nucleobase (HpPNP = 2-hydroxypropyl-4-nitrophenylphosphate), and a uridine containing RNA analog UpPNP (uridine-3′-4-nitrophenylphosphate). Plots of the first-order rate constant for cleavage of HpPNP as a function of Zn(L2) concentration from 0.5 mM to 20.0 mM are linear, consistent with weak complexation to substrate K_d > 20 mM. In contrast, first-order rate constants for cleavage of UpPNP by Zn(L2) or Zn₂(L1) over similar concentration ranges exhibit a downward curvature, consistent with the formation of a complex between catalyst and UpPNP. Comparison of second-order rate constants (k₂ = k_cat/K_d) shows that the dinuclear complex Zn₂(L1) is a better catalyst than Zn(L2) for both HpPNP and UpPNP cleavage.     

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.