Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency

Abstract

Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our reporter construct to investigate if telomeric sequences near an induced double-strand break alter the frequency of recombination between nontelomeric sequences, and found no differences among the different cell lines analysed. Our results indicate that the underlying defect in homologous recombination in ALT cells does not affect sequences independent of their chromosomal location but is likely to be primarily a specific telomeric defect.

Figure 1

Recombination reporter systems. (A) Schematic illustration of the pDR-GFP recombination reporter system (left) and its modification into a chromosomal healing vector (right). HindIII cuts three times in the pDR-GFP recombination reporter system, excising an 812 bp internal band as well as a second band of variable size, depending on the genomic integration site, and therefore allowing determination of copy number. For the chromosomal healing vector, a 1.6 kb telomeric sequence was cloned 482 bp downstream of the iGFP gene. Due to cloning, additional PstI and HindIII sites were introduced into the plasmid giving rise to bands of fixed size following HindIII digestion. In order to increase the possibility of a chromosomal healing event, the vector was linearized with ClaI before transfection. All Southern blots except for those after Bal31 treatment were probed with a HindIII-excised 812 bp GFP probe. (B) Diagnostic algorithm for determination of internal versus subtelomeric position after transfection with the chromosomal healing vector. The pattern of bands expected after digestion with PstI and EcoRV helps to distinguish between one and more than one integrated subtelomeric copy. Since all clones contained a complete cassette of the reporter construct, the lack of a second band is decisive to distinguish between the internal and NextTel position. The left-hand HindIII site is not present in the basic recombination reporter vector shown in (A), so the 2.7 kb band becomes a band of variable size dependent on the integration site.

Figure 2

Molecular analysis for determining the copy number and position of the recombination reporter system. (A) Southern blot analysis of all recombinogenic SW39 and SW26 clones with the reporter system in an intrachromosomal position (20 μg HindIII-digested genomic DNA per lane). (B) EcoRV Southern analysis of SW39 and SW26 clones transfected with the chromosomal healing vector. All clones further analysed for their frequency of recombination had one copy either next to the healed telomere (SW39 #B, #C, #E; SW26 #B, #D, #F, #G) or at an internal location (SW39 #A, #F; SW26 #C, #E). Clones SW39 #D and SW26 #A are examples of clones that were excluded due to multiple copies inserted. (C) PstI Southern analysis of SW39 and SW26 clones after transfection with a chromosomal healing vector. The presence of one single band represents a clone with the recombination reporter system in the NextTel position, whereas two bands indicate an internal+ location. (D) Bal31 digestion of SW26 clones #E and #G as an example for the accuracy of molecular analysis by conventional Southern blotting. In all, 15 μg of genomic DNA was treated with 2.5 U of Bal31 for 1 and 2 h (#E) or for 2 and 4 h (#G), respectively, followed by EcoRI digestion. In contrast to the clone with internal position, the clone with NextTel location of the reporter system shows sensitivity to Bal31 treatment after hybridizing with a Puro-GFP probe. (E) FISH analysis confirmed the data obtained by Southern blot analysis. (Ea) A clone with internal position. In contrast to that, (Eb) a clone with subtelomeric location of the integrated pDR-GFP reporter system is shown.

Figure 3

FACS analysis and calculation of the frequency of recombination after an induced homologous recombination process had occurred. Mock electroporated cells that are negative for GFP expression for most of the clones analysed show autofluorescence and fall along a diagonal between orange (x-axis) and green (y-axis). Some clones exhibit background activity, which never exceeded levels above 0.5%. In contrast, GFP-expressing cells shift towards the green axis. Positive control samples determine the transfection efficiency for each cell clone in each individual experiment. The frequency of recombination is calculated according to the following formula: frequency (%)=((number of GFP positive cells after I Sce-I induction − background)/(number of GFP transfection − background)).