Figure 2.

Down-regulation of DNA ligase I leads to reduced MHEJ activity. HTD114 cells were transfected with siRNA oligonucleotides against DNA ligase I (siRNA 121249). Forty-eight hours after transfection, total RNA and nuclear extracts were prepared. RNA samples were reverse transcribed to cDNA, followed by real time-PCR, repeated three times for each RNA sample, to determine the expression level of ligase I (A). Data represent the mean and the SEM. Nuclear extracts were analyzed by immunoblotting with the indicated antibodies (B). (C) End-joining activity affected by the depletion of Lig1. Linearized pUC18PD1/4 DNA (300 ng) was incubated without (lane 1) or with 1 μg nuclear protein extracts from HTD114 transfected with the indicated siRNA oligonucleotides (lanes 2 and 3). The end-joined products were separated by electrophoresis in an agarose gel and the efficiency of end joining was calculated as the percentage of end-joined products (dimer and multimers) in total DNA in the reaction (monomer, dimer and multimers). (D) A summary of three independent end-joining experiments performed as in C (above). Data represent the mean and SEM. (E) The amount of end-joined products from individual reactions shown in C (above) was determined, in triplicate, with a quantitative real time-PCR assay as described in Materials and methods section. The bar represents the mean and the error bar is SEM. (F) End-joined products from reactions shown in C were PCR amplified and subsequently digested with XcmI. The relative contribution of 10-bp MHEJ to DNA end joining was calculated as the percentage of the XcmI-digested fragments in total PCR products (sum of the undigested and XcmI-digested fragments). (G) Quantification of MHEJ activity. The MHEJ activity was calculated by multiplying the amount of end-joined products measured from (E) with the relative contribution of MHEJ obtained in (F).