Inhibition of the Bloom's and Werner's Syndrome Helicases by G-Quadruplex Interacting Ligands

Li, J. L; Harrison, R. John; Reszka, Anthony P.; Brosh, Robert M.; Bohr, Vilhelm A.; Neidle, Stephen; Hickson, Ian D.

doi:10.1021/bi011067h

Cited by 99 publications

(75 citation statements)

References 58 publications

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“…These molecules preferentially localize and act at telomeres. They inhibit the activity of canonical DNA polymerases (24) and helicases (25,26) on telomeric substrates and delocalize telomeric proteins from telomeres (27), which, together with their effects on telomerase activity, participate to their selective cytotoxicity toward cancer cells and their efficiency as anticancer drugs. Telomerase is one of the enzymes that must be evaluated in the design and development of new G-quadruplex ligands, and we provide here an approach to validate carefully such candidates.…”

Section: Mechanisms Of Action Of G-quadruplex Ligandsmentioning

confidence: 99%

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Cian

Cristofari

Reichenbach

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

268

193

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Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.G-quadruplex ͉ telomere ͉ telomeric repeat amplification protocol assay ͉ direct assay T he unlimited proliferative potential of cancer cells depends on telomere maintenance (1). As a consequence, several classes of telomerase inhibitors have been developed for anticancer purposes. Optimal telomerase activity requires an unfolded single-stranded telomeric overhang (2-4). This overhang may adopt an unusual DNA structure called a G-quadruplex, composed of G-quartets and formed in the presence of cations (5). Therefore, ligands that selectively bind to and stabilize telomeric G-quadruplex structures could act as indirect telomerase inhibitors (Fig. 1A) (6). The number of identified G-quadruplex ligands has grown rapidly over the past few years (for a recent review, see ref. 7); some of these molecules exhibit potent in vivo antitumor activity, and clinical trials started recently.Although various methods are available to measure telomerase activity, the telomeric repeat amplification protocol (TRAP) is most widely used (8). With a few notable exceptions (6, 9-12), TRAP [or a variant called TRAP-G4 (13)] has been the standard method to measure telomerase inhibition by G-quadruplex ligands (reviewed in ref. 7). The original method was improved by the use of an internal control (often called ITAS for internal telomerase assay standard) that coamplifies with the telomerase products and allows the detection of nonspecific Taq polymerase inhibitors. Notably, the ITAS product does not contain any telomeric sequence and therefore is not able to form a G-quadruplex (5,14). We demonstrate here that TRAP is...

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Section: Mechanisms Of Action Of G-quadruplex Ligandsmentioning

confidence: 99%

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Cian

Cristofari

Reichenbach

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

268

193

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“…We therefore examined whether WRN was involved in the CRS assay. As an initial approach, we performed the CRS assay in the presence of a specific inhibitor (tetra-N-methylpyridylporphin) of RecQ helicases (61) and found that this drug was strongly inhibitory (Fig. 6A).…”

Section: The Pso4 Complex Is Required For In Vitro Processing Of Icls-tomentioning

confidence: 99%

The Pso4 mRNA Splicing and DNA Repair Complex Interacts with WRN for Processing of DNA Interstrand Cross-links

Zhang

Kaur

et al. 2005

Journal of Biological Chemistry

110

116

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DNA interstrand cross-links (ICLs) are perhaps the most formidable lesion encountered by the cellular DNA repair machinery, and the elucidation of the process by which they are removed in eukaryotic cells has proved a daunting task. In particular, the early stages of adduct recognition and uncoupling of the cross-link have remained elusive principally because genetic studies have not been highly revealing. We have developed a biochemical assay in which processing of a DNA substrate containing a site-specific psoralen ICL can be monitored in vitro. Using this assay we have shown previously that the mismatch repair factor MutS␤, the nucleotide excision repair heterodimer Ercc1-Xpf, and the replication proteins RPA and PCNA are involved in an early stage of psoralen ICL processing. Here, we report the identification of two additional factors required in the ICL repair process, a previously characterized pre-mRNA splicing complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex), and WRN the protein deficient in Werner syndrome. Analysis of the WRN protein indicates that its DNA helicase function, but not its exonuclease activity, is required for ICL processing in vitro. In addition, we show that WRN and the Pso4 complex interact through a direct physical association between WRN and Cdc5L. A putative model for uncoupling of ICLs in mammalian cells is presented.

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“…These structures could make the damaged D-loops the preferred substrates for BLM and WRN helicase activity. One of the possible structures could be G-quadruplex (G4) formation, as both BLM and WRN work particularly efficiently on these structures (33). Szalai et al (46) reported the formation of G-quadruplex and other secondary structures upon conversion of guanine to 8-oxodG in telomeric DNA in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…This result clearly demonstrates that there is no contaminating glycosylase activity in the proteins used in this study. (33,34) and have been implicated in DNA repair (12,15,16). On the other hand, the role of RecQ5 is not clear yet (35).…”

Section: Construction Of D-loops With and Without The 8-oxodgmentioning

confidence: 99%

Telomeric D-loops Containing 8-Oxo-2′-deoxyguanosine Are Preferred Substrates for Werner and Bloom Syndrome Helicases and Are Bound by POT1

Ghosh

Rossi

Aulds

et al. 2009

Journal of Biological Chemistry

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8-Oxo-2-deoxyguanosine (8-oxodG) is one of the most important oxidative DNA lesions, and G-rich telomeric DNA is especially susceptible to oxidative DNA damage. RecQ helicases WRN and BLM and telomere-binding protein POT1 are thought to play roles in telomere maintenance. This study examines the ability of WRN, BLM, and RecQ5 to unwind and POT1 to bind telomeric D-loops containing 8-oxodG. The results demonstrate that WRN and BLM preferentially unwind telomeric D-loops containing 8-oxodG and that POT1 binds with higher affinity to telomeric D-loops with 8-oxodG but shows no preference for telomeric single-stranded DNA with 8-oxodG. We speculate that telomeric D-loops with 8-oxodG may have a greater tendency to form G-quadruplex DNA structures than telomeric DNA lacking 8-oxodG.Telomeres are structures at the ends of the eukaryotic linear chromosomes that enhance chromosome stability by preventing DNA end-initiated recombination, exonucleolytic attack, and replication-associated terminal recession. Telomeres are composed of long tracts of short tandem repeated DNA sequences (5Ј-TTAGGG-3Ј in human and mouse) and telomere-specific DNA-binding proteins. The length of telomeres is maintained by an active process, and defects in telomere maintenance lead to telomere attrition, genome instability, cell cycle arrest, and senescence or apoptosis. Telomere attrition is frequently associated with aging

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Inhibition of the Bloom's and Werner's Syndrome Helicases by G-Quadruplex Interacting Ligands

Cited by 99 publications

References 58 publications

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

The Pso4 mRNA Splicing and DNA Repair Complex Interacts with WRN for Processing of DNA Interstrand Cross-links

Telomeric D-loops Containing 8-Oxo-2′-deoxyguanosine Are Preferred Substrates for Werner and Bloom Syndrome Helicases and Are Bound by POT1

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