The mechanism of activation of the Alternative Lengthening of Telomeres (ALT) pathway of mammalian chromosome end maintenance has remained an unresolved issue. We have discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT associated PML bodies (APBs), extra-chromosomal telomeric DNA species an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and inter-telomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We identified that ALT induction is positively regulated by RAD17 and BLM, while negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly support the hypothesis that ALT is a consequence of a histone management dysfunction.

The concept of a G-quartet, a unique structural arrangement intrinsic to guanine-rich DNA, was first introduced by Gellert and colleagues over 40 years ago. For decades, it has been uncertain whether the G-quartet and the structure that it gives rise to, the G-quadruplex, are purely in vitro phenomena. Nevertheless, the presence of signature G-rich motifs in the eukaryotic genome, and the plethora of proteins that bind to, modify or resolve this nucleic acid structure in vitro have provided circumstantial evidence for its physiological relevance. More recently, direct visualisation of G-quadruplex DNA at native telomeres was achieved, bolstering the evidence for its existence in the cell. Furthermore, G-quadruplex folded telomeric DNA has been found to perturb telomere function and to impede the action of telomerase, an enzyme overexpressed in >85% of human cancers, hence opening up a novel avenue for cancer therapy in the form of G-quadruplex stabilising agents.

Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Interand intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K þ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na þ -stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter-but not intramolecular K þ -stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase.

Summary Recent evidence for 5’ cytosine (C)-rich overhangs at the telomeres of the nematode C. elegans provided the impetus to re-examine the end structure of mammalian telomeres. Two-dimensional (2D) gel electrophoresis, Single Telomere Length Analysis (STELA) and strand-specific exonuclease assays revealed the presence of a 5’ C-rich overhang at the telomeres of human and mouse chromosomes. C-overhangs were prominent in G1/S arrested as well as terminally differentiated cells, indicating that they did not represent replication intermediates. C-rich overhangs were far more prevalent in tumor cells engaged in the Alternative Lengthening of Telomeres (ALT) pathway of telomere maintenance, which relies on the homologous recombination (HR) machinery. Transient siRNA based depletion of the HR-specific proteins RAD51, RAD52 and XRCC3 resulted in changes in C overhang levels, implicating the involvement of 5’ C-overhangs in the HR-dependent pathway of telomere maintenance.

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