Chromosome ends are marked by the G-rich composition of the strand with 5’-3’ orientation and its protrusion as a single-stranded (ss) DNA tail, known as the G-overhang (Makarov et al., 1997
). The overhang is essential for the formation of a protective higher-order structure referred to as the T-loop (Griffith et al., 1999
), which is postulated to hinder unwanted DNA repair and nuclease activities at the chromosome end; it is the obligate substrate for the ribonucleoprotein enzyme telomerase needed to synthesize telomeric DNA (Greider and Blackburn, 1985
) and is the binding site for POT1 (protection of telomeres 1) (Baumann and Cech, 2001
), the only member of the telomere-specific six-protein shelterin complex that maintains direct contact with the single stranded overhang (de Lange, 2005
). Finally, it is the key structural motif required for homologous recombination.
While the 3’ G-overhang appears to be a universally conserved structural feature at the chromosome end of most species (Oganesian and Karlseder, 2009
), its 5’ C-rich counterpart has so far only been observed in the nematode C. elegans
(Raices et al., 2008
). In worms G- and C-rich telomeric tails are equally abundant and are sequence specifically bound by two C. elegans
OB fold containing proteins, CeOB1 and CeOB2, respectively. Curiously, deletion of CeOB1 yields an overhang and telomere lengthening phenotype, whereas loss of CeOB2 causes telomere length heterogeneity. This suggests that while the G-overhang-specific CeOB1 protein may regulate telomerase, CeOB2 could prevent uncontrolled losses and gains of telomeric DNA through deregulated recombination, which is a defining feature of the ALT phenotype. It is unclear whether the G- and C-overhangs can coexist at the alternative ends of the same chromosome in the worm and whether the latter overhang can also participate in end protection. Nonetheless, in vitro
both CeOB1 and CeOB2 have been demonstrated to localize to the base of T-loops (Raices et al., 2008
). In addition, there is evidence from in vitro
strand invasion assays that artificially constructed 3’ G-rich and 5’ C-rich overhangs of vertebrate telomeric sequences can invade double-stranded DNA with equal efficiency (Verdun and Karlseder, 2006
). Taken together, these observations allow for the speculation that T-loop formation and/or formation of telomeric recombination intermediates may involve the 5’ C-rich telomeric overhang in C. elegans
and other species.
The majority of human tumors rely on telomerase as a strategy to maintain telomere length and to gain immortality. However, approximately 10 % of human cancers negate telomere loss through homologous recombination, employing a telomere length maintenance mechanism known as ALT (Bryan et al., 1997
). Tumor cells engaged in this pathway exhibit a number of distinctive characteristics (Henson et al., 2009
): pronounced heterogeneity of telomere length, presence of extra-chromosomal telomeric repeats (ECTR) of both linear and circular conformations, including C-rich ss circles (C-circles), occurrence of sub-nuclear foci known as ALT-associated PML bodies (APBs) wherein PML protein co-localizes with telomeric DNA and proteins involved in telomere maintenance, DNA repair and HR and, lastly, a high incidence of telomere sister chromatid exchange (t-SCE) events.
The recombination process in ALT is thought to proceed via either inter- or intramolecular telomeric invasion. The former was demonstrated when telomeres tagged with a selection marker could pass on their tag to neighboring telomeres with increasing passage in culture (Dunham et al., 2000
). No tag movement was observed in telomerase positive cells. There is now also evidence that supports recombination through intramolecular self-invasion, demonstrating that an individual telomere can use itself as a template to initiate telomeric DNA synthesis (Muntoni et al., 2009
An intricate interplay of recombination proteins facilitates DNA repair and synthesis through homologous recombination. Three of the key players in this process are the RAD51 recombinase, the RAD52 recombination-mediator and XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3), one of the five RAD51 paralogs. The core function of RAD51 is to promote strand invasion during the early stages of HR, which is achieved via polymerization of this recombinase onto a 3’ ss DNA end and mediating the transfer and annealing of the resulting nucleoprotein filament to a complementary homologous strand (Sung and Klein, 2006
). RAD52 stimulates RAD51-mediated strand invasion and enhances its specificity for ss DNA binding via direct interaction with RAD51 in yeast (Benson et al., 1998
; New et al., 1998
; Shinohara and Ogawa, 1998
; Sung, 1997
), and this protein also exhibits ss DNA binding ability and promotes single-strand annealing (SSA) reactions independently of RAD51 (Mortensen et al., 1996
). XRCC3 has been implicated in both early and late HR events, namely facilitating the assembly of the active RAD51 nucleoprotein filament on the ss overhang (Forget et al., 2004
), a function that it shares with RAD52, and performing Holiday Junction resolution via endonucleolytic cleavage of the strands participating in strand invasion and D-loop formation (Liu et al., 2004
). It has been proposed that the base of the T-loop may serve as a substrate for XRCC3 since it closely resembles a Holliday Junction (Wang et al., 2004
). Thus the large abundance of circular ECTR in ALT cells has been partly attributed to XRCC3 (Compton et al., 2007
) and is thought to arise as a result of deregulated recombination.
The molecular mechanism that drives HR-dependent telomere maintenance in ALT remains poorly understood; the substrates, intermediates and products/byproducts of recombination in this pathway are ill defined. To date, the presence of single stranded circles comprised of C rich telomeric DNA are the best indicator for an active ALT mechanism (Henson et al., 2009
), however, it is unclear how such circles are generated. Here we present evidence for the presence of a 5’ C-rich overhang, a structural motif at mammalian telomeres, which is specifically enriched in human ALT cells as well as mouse cells. We find that telomeric C-overhang levels are greatly perturbed in response to depletion of three key HR proteins, RAD51, RAD52 and XRCC3 in ALT cells. Based on these findings we propose that the C-rich overhang is an outcome and driver of HR-dependent telomere maintenance.