We report the first G-quadruplex structure formed in the promoter region of the human bcl-2. Bcl-2 is a potent oncoprotein that functions as an inhibitor of cell apoptosis and has been found to be aberrantly overexpressed in a wide range of human tumors. A highly GC-rich region upstream of the P1 promoter plays an important role in the regulation of the transcriptional activity of the bcl-2 oncogene. The purine-rich strand of this region contains multiple runs of guanines and can form three distinct intramolecular G-quadruplexes in K + -containing solution. Of these, the G-quadruplex formed within the middle four consecutive guanine runs has been shown to be the most stable Gquadruplex structure, while it is also a mixture of loop isomers. This predominate G-quadruplex structure formed in this region was studied by NMR. Our results demonstrate a novel folding of a Email: yangd@pharmacy.arizona.edu. unique intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands. This Gquadruplex structure contains three G-tetrads connected with a single-nucleotide double-chainreversal side loop and two lateral loops. The three-nucleotide CGC loop in the bcl-2 promoter sequence forms a lateral loop, as opposed to a double-chain-reversal side loop observed in a similar sequence in the c-MYC promoter, which appears to largely determine the overall folding of the bcl-2 G-quadruplex. Furthermore, both the bcl-2 and c-MYC promoter sequences contain the G 3 NG 3 sequence motif, which forms a stable double-chain-reversal, parallel-stranded structural motif. This predominant bcl-2 G-quadruplex represents an attractive novel target for the design of new anticancer drugs that specifically modulate bcl-2 gene expression. NIH Public AccessBcl-2 (B-cell CLL/lymphoma 2) is a potent oncoprotein that plays an essential role in cell survival and functions as an inhibitor of cell apoptosis. 1 The bcl-2 proto-oncogene was first discovered in human follicular lymphoma and has been mapped to chromosome 18q21 based on a t(14;18) translocation to the immunoglobulin heavy chain (IgH) locus at 14q32. 2 Bcl-2 has been found to be aberrantly overexpressed in a wide range of human tumors, including Bcell and T-cell lymphomas, breast, prostate, cervical, colorectal, sand non-small cell lung carcinomas. 3 Elevation of bcl-2 level has also been associated with poor prognosis. 3 Thus the bcl-2 transcriptional control has emerged as an attractive target for anticancer therapeutics.The P1 promoter located 1386-1423 base pairs upstream of the translation start site is the major transcriptional promoter for bcl-2. 4 This is a TATA-less, GC-rich promoter that contains multiple transcriptional start sites. The 5′-end of the P1 promoter, including a highly GC-rich region, has been implicated in playing a major role in the regulation of bcl-2 transcription. 4 This GC-rich element is a 39-base-pair sequence that is located 58 to 19 base pairs upstream of the P1 promoter. Deletion or mutation of this element has been shown to increase prom...

The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular Gquadruplex structures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNA sequences within this element that formed the three individual G-quadruplexes. Each of these was characterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The central G-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of three tetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agents were found to further stabilize these structures, with individual selectivity toward one or more of these G-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-myc promoter in that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexity of the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. These results are discussed in relation to the biological significance of this G-quadruplex-forming element in modulation of bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes and loop isomers are possible.

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

Background: Tdp1 is a DNA repair enzyme conserved across eukaryotes. Results: Tdp1 repairs not only 3Ј-tyrosyl-DNA bonds and 3Ј-phosphoglycolates but also 5Ј-tyrosyl-DNA bonds and 3Ј-deoxyribose phosphates. Conclusion:The end processing functions of Tdp1 extend to the repair of Top2-DNA adducts and DNA breaks from base alkylation. Significance: Tdp1 has a broad range of DNA repair activities and is a potential drug target in anticancer therapy.