1. Mammalian 5′ C-Rich Telomeric Overhangs Are a Mark of Recombination-Dependent Telomere Maintenance 
Liana Oganesian, Jan Karlseder 
Molecular Cell 
Volume 42, Issue 2, Pages (April 2011)
DOI: /j.molcel
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2. Figure 1
Cytosine-Rich Telomeric Overhangs at Human and Mouse Chromosome Ends
(A) Schematic representation of 2D gel electrophoresis whereby DNA is separated according to its molecular weight (one-dimensional [1D]) and conformation (2D). Native electrophoresis allows for visualization of DNA overhangs and extrachromosomal SS telomeric material (left panel), whereas denaturing conditions establish the presence of DS telomeric DNA of linear or circular conformations (right panel).
(B) Restriction-digested genomic DNA from human KMST6, Saos2, U2OS, IMR90, HeLa, and mouse (MEFs) cell lines was electrophoresed in two dimensions and probed for DNA of G-rich (top panels) or C-rich (bottom panels) telomeric sequence under native conditions. The high-molecular weight signals of U2OS and mouse telomeres have been indicated. The ratio of the levels of G- to C-overhangs is shown below as an indication of C-overhang enrichment in ALT and mouse cells. Signal intensities corresponding to each overhang were normalized against the signal for their corresponding denatured controls and then compared to each other.
(C) Same procedure as in (B) except that the gels were denatured before hybridization.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 The C-Rich Overhang Is Terminal and 5′ in Orientation
(A) Genomic DNA from Saos2 cells was treated with a 5′-3′ (RecJf) or a 3′-5′ (Exo1) exonuclease, followed by a restriction digest. A 1D image of nuclease-treated DNAs (20 μg) loaded onto an agarose gel and stained with ethidium bromide serves as a loading control.
(B) Lanes corresponding to each DNA sample from (A) were excised and electrophoresed in the second dimension and in-gel hybridizations with strand-specific 32P end-labeled oligonucleotide probes were performed to demonstrate DNA of G-rich (top panels) or C-rich (bottom panels) telomeric sequence under native conditions.
(C) Same procedure as in (B), except that the gels in (B) were denatured before rehybridization.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
The Telomeric C-Rich Overhang Persists across the Cell Cycle and Is Present in Terminally Differentiated Cells
(A) Saos2 human tumor cells were synchronized by the thymidine/aphidicolin double block method and collected and processed for native (left panels) and denaturing (right panels) 2D gel analyses at 0, 4, 8, and 12 hr after release from cell-cycle arrest. Corresponding cell-cycle phases are indicated on the left and the cell-cycle distribution (in %) is indicated in the right panel. The ratio of native to denatured signal was normalized to this ratio in asynchronous cells and is indicated in the left panels. Ethidium bromide staining of the first dimension is shown for loading accuracy (bottom panel).
(B) C2C12 mouse muscle cell precursors or myoblasts were induced to differentiate into muscle myotubes by introducing 2% horse serum in place of fetal bovine serum in the growth medium. Genomic DNA (25 μg) either from myoblasts (top panels) or myotubes (bottom panels) was extracted, restriction-digested, and processed for 2D gel analysis under native and denaturing conditions. The high-molecular weight telomeric signal is indicated. Ethidium bromide staining of the first dimension of each gel is shown for loading accuracy (bottom panel).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The Telomeric C-Overhang Is at Least Partly of Chromosomal Origin, but Maintains Close Association with the Extrachromosomal Telomeric Compartment in ALT Cells
(A) Schematic representation of the arcs resulting from native 2D gel electrophoresis of telomeric DNA from Saos2 Hirt extracts. Native electrophoresis allows for visualization of a DNA overhang (open arrowheads) and extrachromosomal SS telomeric material, including nicked or gapped DS circles (black arrowheads), SS circles, and linear DNA fragments (gray arrowheads).
(B) Digested and undigested DNAs from the supernatant (Sup) and the accompanying pellet fraction of Hirt lysate preparations from Saos2 cells were separated by standard agarose gel electrophoresis in the first dimension and stained with ethidium bromide to determine equal loading. The molecular weight (MW) marker on the left is the 1 kb DNA ladder from Invitrogen with MW range of 500 bp to 12 kb.
(C) Undigested (left panels), AluI and MboI-digested Hirt extract Sup fractions (middle panels), and AluI and MboI-digested pellet fractions (right panels) were hybridized with probes specific for the telomeric C or G strand under native (top two panels) or denaturing (bottom two panels) conditions.
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6. Figure 5
RAD51 Knockdown Increases the Prevalence of the C- but Not G-Rich Telomeric Overhangs
(A) Immunoblot with antibodies against RAD51 from KMST6 whole-cell extracts prepared 72 hr after transfection with control or RAD51-specific siRNAs. Tubulin was used as a loading control.
(B) Ethidium bromide staining of the first dimension of the 2D gel electrophoresis. Twenty micrograms of DNA was loaded per lane.
(C) Each lane in (B), top panel, was excised, electrophoresed in the second dimension, and in-gel hybridized with 32P end-labeled (TTAGGG)4 oligonucleotide probe specific for the C strands of telomeric DNA under native and denaturing conditions. Treatment with control and RAD51-specific siRNAs is indicated.
(D) Each lane in (B), bottom panel, was excised, electrophoresed in the second dimension, and in-gel hybridized with 32P end-labeled (CCCTAA)5 oligonucleotide probe specific for the G strands of telomeric DNA under native and denaturing conditions. Treatment with control and RAD51-specific siRNAs is indicated.
(E) Quantification of three to four independent experiments of RAD51 targeting. The ratio between native and denatured signals is indicated in arbitrary units. The error bars represent standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
RAD52 Knockdown Increases the Prevalence of C- but Not G-Rich Telomeric Overhangs
(A) Immunoblot with antibodies against RAD52 from KMST6 whole-cell extracts prepared 72 hr after transfection with control or RAD52-specific siRNAs. Tubulin was used as a loading control.
(B) Ethidium bromide staining of the first dimension of the 2D gel electrophoresis. Twenty micrograms of DNA was loaded per lane.
(C) Each lane in (B), top panel, was excised, electrophoresed in the second dimension, and in-gel hybridized with 32P end-labeled (TTAGGG)4 oligonucleotide probe specific for the C strands of telomeric DNA under native and denaturing conditions. Treatment with control and RAD52-specific siRNAs is indicated.
(D) Each lane in (B), bottom panel, was excised, electrophoresed in the second dimension and in-gel hybridized with 32P end-labeled (CCCTAA)5 oligonucleotide probe specific for the G strands of telomeric DNA under native and denaturing conditions. Treatment with control and RAD52-specific siRNAs is indicated.
(E) Quantification of overhang levels upon RAD52 depletion shown as the ratio between native and denatured signals and indicated in arbitrary units. The error bars represent standard deviation of three independent experiments.
(F) Native dot blot of the G-rich linear telomeric products of the phi29-dependent C-circle assay. Twenty-five to two hundred fifty nanograms of restriction-digested genomic DNA from control or RAD52-depleted KMST6 cells was used in each assay. The blot was hybridized with 32P end-labeled (CCCTAA)5 oligonucleotide probe.
(G) Quantification of dot blot signal in (F) expressed in arbitrary units.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Model for the Possible Genesis and Maintenance of 5′-C-Overhangs at Human Telomeres that Are Prone to Recombination
(i and ii) In the context of telomere dysfunction (i) intertelomeric recombination (ii) may be initiated through 3′ G-overhang-mediated invasion of neighboring chromosome ends, which are then used as a template for subsequent DNA synthesis.
(iii–v) Alternatively, given sufficient length, a looped and partially protected conformation (iii) may be adopted, leading to an XRCC3- and NBS1-dependent resolution event (Wang et al., 2004) thought to give rise to extrachromosomal DS nicked T-circles (iv) and truncated telomeres (v). The precise mechanism of T-loop cleavage is unknown allowing for the possibility that the end structure of the resultant telomere may not necessarily contain the conventional 3′ G-overhang but rather a 5′-C-rich counterpart (v). The telomere bearing a 5′ overhang may be less stable than its G-rich equivalent because there is no known human protein that has an affinity specifically for SS C-rich telomeric DNA.
(vi and vii) If sufficiently long it may self-invade to form a small T loop with an exposed SS displacement loop (vi), perhaps making it susceptible to nuclease attack. It is conceivable that this C-overhang-mediated loop formation may be the precursor for C-circles (vii).
(viii–x) Participation of these in RCA (viii) with accompanying lagging strand synthesis could give rise to a product that is a linear DS extrachromosomal telomeric array (ix). There is precedent for an RCA-dependent mode of telomere maintenance at mitochondria of C. parapsilosis (Nosek et al., 2005). In this organism the RCA product bears a 5′ overhang (ix), which allows for invasion into and recombination with chromosome ends (x) endowing them with a 5′ telomeric overhang (v) (Nosek et al., 1995, 2005; Tomaska et al., 2009). We suggest that this could occur at telomeres of human ALT cells, given the prevalence of 5′-C-overhangs in these cells. Because T-loop formation can be facilitated by 5′ overhang-mediated self-invasion (vi) in C. parapsilosis (Nosek et al., 2005) and also in the worm (Raices et al., 2008), the existence of such a structure in human cells is conceivable. In the context of diminished RAD51 levels, G-overhangs of short dysfunctional telomeres that are subject to recombination may be at a risk of degradation via the action of unknown 3′-5′ exonucleases (xi) further contributing to C-overhang formation (v). This also holds true for XRCC3 and RAD52, probably because these are involved in recruitment of RAD51 to overhangs and stimulating its strand transferase activity, respectively. Depletion of RAD52 results in an increase in C-circle levels (Figures 6F and 6G), supporting the notion that C-circles are generated upstream of RAD52 action and are a byproduct of 5′ overhang-mediated intratelomeric recombination and/or a substrate for RCA. The 2D gel schematic on the right shows the arcs representing the DNA species discussed.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions