Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.

RNF126 is an E3 ubiquitin ligase. The deletion of RNF126 gene was observed in a wide range of human cancers and is correlated with improved disease-free and overall survival. These data highlights the clinical relevance of RNF126 in tumorigenesis and cancer therapy. However, the specific functions of RNF126 remain largely unknown. Homologous recombination (HR)-mediated DNA double-strand break repair is important for tumor suppression and cancer therapy resistance. Here, we demonstrate that RNF126 facilitates HR by promoting the expression of BRCA1, in a manner independent of its E3 ligase activity but depending on E2F1, a well-known transcription factor of BRCA1 promoter. In support of this result, RNF126 promotes transactivation of BRCA1 promoter by directly binding to E2F1. Most importantly, an RNF126 mutant lacking 11 amino acids that is responsible for the interaction with E2F1 has a dominant-negative effect on BRCA1 expression and HR by suppressing E2F1-mediated transactivation of BRCA1 promoter and blocking the enrichment of E2F1 on BRCA1 promoter. Lastly, RNF126 depletion leads to the increased sensitivity to ionizing radiation (IR) and poly (ADP-ribose) polymerase (PARP) inhibition. Collectively, our results suggest a novel role of RNF126 in promoting HR-mediated repair through positive regulation on BRCA1 expression by direct interaction with E2F1. This study not only offers novel insights into our current understanding of the biological functions of RNF126 but also provides a potential therapeutic target for cancer treatment.

Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells.

Small interfering RNA (siRNA) is normally designed to silence preselected known genes. Such selections are inevitably prone to bias as a result of limited knowledge about the biological process, transcript identity, and functions. A library that contains all permutations of siRNA could avoid such problems. In this paper, it is shown that 5 ؋ 10 7 siRNA-encoding plasmids can be constructed in a single tube by using vectors with two mutated RNA polymerase III promoters arranged in a convergent manner. Such a library was used to carry out genomewide screening of functional genes in a phenotype-driven manner. Multiple siRNAs that induce a significant increase of cell proliferation speed were identified.random targeting ͉ screening ͉ vector ͉ dual promoters T he discovery of the RNA interference (RNAi) phenomena, especially small interfering RNA (siRNA), has dramatically simplified the manipulation of gene expression for functional genomics and drug target validation (1-3). Synthetic siRNAs were introduced and have been widely used for gene knockdown experiments (2, 3). siRNA-expressing plasmid or viral vectors were subsequently shown to be very effective in down-regulation of target mRNA and protein levels (4-10). The rapid development of this powerful method has made it possible to knock down almost any known gene in an applicable organism. The application of the method is, however, restricted by the required knowledge of the sequence of a gene to create siRNA to target it. In organisms where the transcriptome is largely known (such as human and mouse), the limitation on large-scale application is mostly reflected on the throughput level, which is further translated into limitations on cost and speed levels. As a consequence, most researchers are forced to shorten their gene list, either by knowledge-based prioritization or by using other first-line high-throughout methods such as microarrays to fit the research into individual cost structure and time frame. In important model species for which the transcriptome is largely unknown, parallel high-throughput applications are practically not possible. To overcome these limitations, the concept of siRNA libraries has been explored.Here we present the construction of siRNA libraries that contain all permutations of siRNA sequences. For this purpose we created a plasmid vector system that contains two convergent RNA polymerase III (Pol III) promoters to drive the expression of both strands of the siRNA from a single randomized region in living cells. We further demonstrated the usefulness of this type of siRNA library for phenotype-driven genomewide screening in a cell proliferation model. Materials and Methods Construction and Validation of Vectors with Dual Pol III Promoters.The pBluescript vector with a human H1 promoter was a kind gift from Mauro D'Amato (Karolinska Institutet). The plasmid with a U6 promoter, pAV͞U6, was a kind gift from David Engelke (University of Michigan, Ann Arbor) (4). First, the H1 promoter was engineered to replace the last 11 nucleotide pai...