A recent study using a quantitative fluorescence titration technique showed that Pol X binds gapped DNA in two distinct modes: one utilizing the total DNA binding site of Pol X and the other utilizing only the auxiliary DNA binding site where the enzyme binds to the double-stranded region of the DNA (28).
Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends.
For instance, TRF1 binds to the double-stranded TTAGGG/CCCTAA repeats, TRF2 binds to the junction of the double-stranded and single-stranded telomeric region, and POT1 binds to single-stranded telomeric repeats (G-tails).
Glutathione-S-transferase P1 expression was determined by real-time PCR on a LightCycler instrument (Roche Diagnostics, Meylan, France) using a SYBRGreen® fluorescent dye, which binds to the double-stranded DNA yielding fluorescence.
The WT MeCP2 fragment binds to the double-stranded DNA probe with increasing amount of protein, but the R306C mutation completely abolished the ability of the fragment to bind DNA.
The Quant-iT PicoGreen reagent, which binds to the double-stranded DNA in solution, was then added, and fluorescence intensity was recorded using a FLx800 microplate fluorescence reader (BioTek, Potton, UK) using 485-nm excitation and 520-nm emission.
In yeast, the Rap1/ Rif1/ Rif2 protein complex binds to the double-stranded telomere repeats (Longtine et al. 1989; Hardy et al. 1992; Wotton and Shore 1997), and the Cdc13 complex (Lin and Zakian 1996; Nugent et al. 1996) binds to the single-stranded G-rich 3′ overhang.
TRF1 and TRF2 bind to the double stranded T-loop of telomeric DNA and are implicated in maintaining the formation of the T-loop structure, while POT1 interacts with single-stranded TTAGGG repeats at the 3′ overhang, as well as in the D-loop of the T-loop configuration.
The proteins involved in the shelterin complex include TRF (telomeric repeat-binding factor) 1 and TRF2, which bind to the double-stranded segment of telomeric DNA [ 9, 10].
As expected in this competition experiment, Phen-DC3 did not bind to the double-stranded DNA competitor since the T1/2 was not affected by increasing concentrations of this duplex (Fig. 2D,F).
