DNA Translocation by Human Uracil DNA Glycosylase: Role of DNA Phosphate Charge

Human DNA repair glycosylases must encounter and inspect each DNA base in the genome to discover damaged bases that may be present at a density of <1 in 10 million normal base pairs. This remarkable example of specific molecular recognition requires a reduced dimensionality search process (facilitated diffusion) that involves both hopping and sliding along the DNA chain. Despite the widely accepted importance of facilitated diffusion in protein–DNA interactions, the molecular features of DNA that influence hopping and sliding are poorly understood. Here we explore the role of the charged DNA phosphate backbone in sliding and hopping by human uracil DNA glycosylase (hUNG), which is an exemplar that efficiently locates rare uracil bases in both double-stranded DNA and single-stranded DNA. Substitution of neutral methylphosphonate groups for anionic DNA phosphate groups weakened nonspecific DNA binding affinity by 0.4–0.5 kcal/mol per substitution. In contrast, sliding of hUNG between uracil sites embedded in duplex and single-stranded DNA substrates persisted unabated when multiple methylphosphonate linkages were inserted between the sites. Thus, a continuous phosphodiester backbone negative charge is not essential for sliding over nonspecific DNA binding sites. We consider several alternative mechanisms for these results. A model consistent with previous structural and nuclear magnetic resonance dynamic results invokes the presence of open and closed conformational states of hUNG. The open state is short-lived and has weak or nonexistent interactions with the DNA backbone that are conducive for sliding, and the populated closed state has stronger interactions with the phosphate backbone. These data suggest that the fleeting sliding form of hUNG is a distinct weakly interacting state that facilitates rapid movement along the DNA chain and resembles the transition state for DNA dissociation

Arsenicals, the Integrated Stress Response, and Epstein–Barr Virus Lytic Gene Expression

Following our observation that clofoctol led to Epstein–Barr virus (EBV) lytic gene expression upon activation of the integrated stress response (ISR), we decided to investigate the impact of As2O3 on viral lytic gene expression. As2O3 has also been reported to activate the ISR pathway by its activation of the heme-regulated inhibitor (HRI). Our investigations show that As2O3 treatment leads to eIF2α phosphorylation, upregulation of ATF4 and TRB3 expression, and an increase of EBV Zta gene expression in lymphoid tumor cell lines as well as in naturally infected epithelial cancer cell lines. However, late lytic gene expression and virion production were blocked after arsenic treatment. In comparison, a small molecule HRI activator also led to increased Zta expression but did not block late lytic gene expression, suggesting that As2O3 effects on EBV gene expression are also mediated through other pathways