Transcript-RNA-templated DNA recombination and repair.

Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

RAD52: Paradigm of Synthetic Lethality and New Developments.

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52's biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.

Impact of monopolar TURP, bipolar TURP and photoselective vaporization of prostate for enlarged prostate on erectile function.

OBJECTIVES: To compare monopolar transurethral resection of the prostate (TURP), bipolar TURP and photoselective vaporization of the prostate (PVP) by 120-W GreenLight laser with regard to the impact on International Index of Erectile Function (IIEF)-5 score in patients presenting with lower urinary tract symptoms (LUTS) secondary to prostate >80 mL. METHODS: Between April 2012 and March 2015, 110 patients who satisfied eligibility criteria were divided into three groups according to surgical modality adopted to treat benign prostatic enlargement. Preoperative, perioperative, and follow-up data were collected. The three groups were as follows: group A, monopolar TURP; group B, bipolar TURP; and group C, PVP. RESULTS: The baseline characteristics of the three groups were similar. All the perioperative parameters were significantly favorable in group C compared with the other two groups, except for mean operative time, which was significantly higher in group C. International Prostate Symptom Score, postvoid residual urine, maximum flow rate and quality of life score had significant and similar improvement during follow up in all three groups. Also, prostate volume reduced significantly in all three groups following surgery, but it remained significantly higher in group C patients compared with groups A and B. Mean IIEF-5 score was similar between the three groups at baseline and during each of the follow-up visits. Groups A, B and C had declines of 3.27% (P = 0.34), 2.68% (P = 0.40) and 3.36% (P = 0.35), respectively, in mean IIEF-5 score at 12-month follow up compared with baseline. CONCLUSIONS: Monopolar TURP, bipolar TURP and PVP by 120-W GreenLight laser for prostate size >80 mL do not have a significant impact on IIEF-5 score at 12-month follow up.