SummaryIonizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of cH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage-and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.

Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3 ؊/؊ embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3 ؊/؊ cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3 ؊/؊ embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3 ؊/؊ embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3 ؊/؊ embryos. The inner cell mass of cultured Rev3 ؊/؊ blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase , double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase in translesion synthesis of endogenously and exogenously induced DNA lesions.In cancer genetics, the paradigm holds the sequential mutation of a series of oncogenes and tumor suppressor genes responsible for the evolutionary development of a normal cell into a fully malignant, metastasizing tumor (67). Most mutations are induced by nucleotide damage, originating from endogenous sources or inflicted by exogenous agents (49). Nucleotide damage that is not removed by DNA repair proteins generally leads to an arrest of the replication fork, due to the rigidity of the replicative polymerases, preventing incorporation of a nucleotide opposite a damaged template (51). To escape this arrest, cells possess multiple pathways that enable the completion of DNA replication despite the presence of replication-blocking DNA damage (3, 9).Considerable progress has recently been made in identifying the actors in translesion synthesis, a pathway implicated in replicating damaged DNA. In both prokaryotes and eukaryotes, multiple polymerases have been identified that are capable of replicating DNA templates containing a variety of lesions. This enables the completion of replication and therefore safeguards cellular survival, albeit frequently at the expense of the introduction of mutations. Based on sequence homology and activity in vitro, most of the polymerases associated with translesion synthesis belong to the newly recognized Y superfamily of DNA polymerases (16,21,30,32,57,70). The heterodimeric Saccharomyces cerevisiae polymerase , comprised of the REV3 catalytic subun...

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS–induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS.