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. Author manuscript; available in PMC: 2010 Jul 10.
Published in final edited form as: Cell. 2009 Jul 10;138(1):90–103. doi: 10.1016/j.cell.2009.06.021

Figure 5. Deletion of TRF1 diminishes the replication efficiency of telomeric DNA.

Figure 5

SMARD assay results from three independent experiments in which TRF1 was deleted from TRF1F/F MEFs. Cells were labeled with IdU and CldU as indicated on the right at day 4 after infection with H&R Cre retrovirus (+Cre) or vector control (− Cre). In the upper panel (experiments 1 and 2), DNA was digested with frequently cutting enzymes and telomeric restriction fragments >25 kb were isolated (schematic on the left). Telomeric DNA molecules were identified by FISH and the % of molecules containing IdU and/or CldU was determined. In the middle panel, telomeric MboI/AluI fragments in the 130–180 kb range (see genomic blot inset) were isolated from a CHEF gel and the fraction of telomeric molecules that contained IdU and/or CldU was determined as above. SMARD assay was done in one experiment in which TRF2 was deleted from TRF2F/F DNA-Lig4−/− cells and the fraction IdU and/or CldU labeled telomeric molecules (130–180 kb range) was analyzed. In the lower panel, the DNA preparation of TRF1F/F MEFs used in the middle panel was digested with SwaI and a 180 kb restriction fragment from the Igh locus was isolated. DNA probes from that locus (see map below) were used to identify the Igh fragments on stretched DNA and the ratio of labeled vs. unlabeled fragments was determined.