Results Determination of the Rad51 core promoter region Previous attempts to define the minimal Rad51 promoter have yielded conflicting results and were performed only in a single osteosarcoma cell line, U2-OS [22,23]. In order to better assess the differential expression of the Rad51promoter, we generated a panel of truncated Rad51 promoter mutants (Figure 1), inserted them upstream of a promoterless luciferase reporter and produced a series of replication-defective, E1-deleted Ad5 vectors that were evaluated in a panel of normal and cancer cell lines (Table 1). As can be seen in Figure 2, maximal promoter strength was retained by a small DNA region surrounding the transcription start site (2230/+217). Luciferase activity in cells transduced with a vector containing this element (Rad51core-luc) was essentially indistinguishable from that in cells transduced with vectors containing larger fragments of the Rad51 promoter (Rad51-F6- luc, 22931/+217; Rad51-D230-luc, 2230/+3564) or the intact full-length Rad51 promoter (22931/+3564) (Figure 2A). To assess the selectivity of the promoter elements for the various cell lines, the luciferase activities for each cell line were normalized to promoter activity in normal human lung fibroblasts, Wi-38 cells, which was arbitrarily assigned a value of 1. This analysis Rad51 Targeted Adenoviral Vector (Figure 2B) confirmed that maximal promoter activity and promoter selectivity were conferred by the Rad51 core promoter element (2230/+217). In contrast, the same analysis performed with a CMV promoter showed little to no tumor specificity. In order to assess whether Rad51core promoter activity was correlated with endogenous cellular expression of Rad51 protein, nuclear extracts from each cell type were isolated and analyzed by Western blot for Rad51 protein (Information S1). A non- parametic statistical comparison of Rad51 protein content to Rad51core promoter activity in each cell line revealed a statistically significant correlation (Spearman rank correlation coefficient = 0.7, with a p-value = 0.04; Information S1). To test whether this core element could be refined further, we constructed a series of truncated derivatives of the Rad51core element (Figure 1C). Analysis of the transcriptional activity of these constructs revealed that all of them were significantly less active than the Rad51core element (Figure 3). We therefore focused subsequent studies on the Rad51core promoter construct. Rad51 promoter activity is suppressed by p53 During the course of testing the Rad51 promoter truncations, we observed that the Rad51 promoter appeared to be more active in cells lacking functional p53, than in cells with intact p53 Figure 1. Rad51 promoter constructs. (A) Diagram of the Rad51 gene and upstream region. All labeled positions are specified relative to the transcription start site. (B) Diagram of the different Rad51 promoter truncations. (C) Diagram of the truncations of the Rad51 core region. A putative p53 binding region has been reported at position 2161 to 2117. (D) Diagram of promoter mutations affecting the p53-binding region. doi:10.1371/journal.pone.0028714.g001 PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28714