SNM1A is crucial for efficient repair of complex DNA breaks in human cells.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes.

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of RPA2.

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.

Daughter-strand gaps in DNA replication - substrates of lesion processing and initiators of distress signalling.

Dealing with DNA lesions during genome replication is particularly challenging because damaged replication templates interfere with the progression of the replicative DNA polymerases and thereby endanger the stability of the replisome. A variety of mechanisms for the recovery of replication forks exist, but both bacteria and eukaryotic cells also have the option of continuing replication downstream of the lesion, leaving behind a daughter-strand gap in the newly synthesized DNA. In this review, we address the significance of these single-stranded DNA structures as sites of DNA damage sensing and processing at a distance from ongoing genome replication. We describe the factors controlling the emergence of daughter-strand gaps from stalled replication intermediates, the benefits and risks of their expansion and repair via translesion synthesis or recombination-mediated template switching, and the mechanisms by which they activate local as well as global replication stress signals. Our growing understanding of daughter-strand gaps not only identifies them as targets of fundamental genome maintenance mechanisms, but also suggests that proper control over their activities has important practical implications for treatment strategies and resistance mechanisms in cancer therapy.

RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids.

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance.

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.

New insights into replication clamp unloading.

The sliding clamp protein proliferating cell nuclear antigen (PCNA) is situated at the core of the eukaryotic replisome, where it acts as an interaction scaffold for numerous replication and repair factors and coordinates DNA transactions ranging from Okazaki fragment maturation to chromatin assembly and mismatch repair. PCNA is loaded onto DNA by a dedicated complex, the replication factor C, whose mechanism has been studied in detail. Until recently, however, it was unclear how PCNA is removed from DNA upon completion of DNA synthesis. Two complementary studies now present data strongly implicating the replication factor C-like complex, Elg1/ATAD5-RLC, in the unloading of PCNA during replication in yeast and human cells. They indicate that an appropriate control over PCNA's residence on the chromatin is important for maintaining genome stability. At the same time, they suggest that the interaction of Elg1/ATAD5 with SUMO, which was also reported to contribute to its role in genome maintenance, affects aspects of its function distinct from its unloading activity.

The ubiquitin-proteasome system of Saccharomyces cerevisiae.

Protein modifications provide cells with exquisite temporal and spatial control of protein function. Ubiquitin is among the most important modifiers, serving both to target hundreds of proteins for rapid degradation by the proteasome, and as a dynamic signaling agent that regulates the function of covalently bound proteins. The diverse effects of ubiquitylation reflect the assembly of structurally distinct ubiquitin chains on target proteins. The resulting ubiquitin code is interpreted by an extensive family of ubiquitin receptors. Here we review the components of this regulatory network and its effects throughout the cell.

Timing and spacing of ubiquitin-dependent DNA damage bypass.

During its duplication, DNA, the carrier of our genetic information, is particularly vulnerable to decay, and the capacity of cells to deal with replication stress has been recognised as a major factor protecting us from genome instability and cancer. One of the major pathways controlling the bypass of DNA lesions during replication is activated by ubiquitylation of the sliding clamp, PCNA. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required mainly for an error-free pathway that likely involves template switching. This review is focussed on our understanding of the timing of damage bypass during the cell cycle and the question of how it is coordinated with the progression of replication forks.

SUMO modification of PCNA is controlled by DNA.

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.

SUMO teams up with ubiquitin to manage hypoxia.

Hypoxia, a condition of severe oxygen shortage, induces a transcriptional response that is important for both normal embryonic development and the growth of tumors. In this issue, Cheng et al. (2007) describe how the stability of the major transcriptional regulator of the hypoxic response, HIF1alpha, is modulated by the interplay between the ubiquitin and the SUMO conjugation systems.

Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage.

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.

The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner.

Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that--by means of independent enzymatic activities inherent in its RING and ATPase domains--plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions.

Protein-protein interactions within an E2-RING finger complex. Implications for ubiquitin-dependent DNA damage repair.

The RING finger protein RAD5 interacts and cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in postreplication DNA damage repair in yeast. Previous observations implied that the function of UBC13 and MMS2 is dependent on the presence of RAD5, suggesting that the RING finger protein might act as a ubiquitin-protein ligase specific for the UBC13-MMS2 complex. In support of this notion it is shown here that the contact surfaces between the RAD5 RING domain and UBC13 correspond to those found in other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein ligases. Mutations that compromise the protein-protein interactions either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in vivo that strongly depended on the expression levels of the corresponding mutants. Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting. Finally, this study demonstrates that beyond its cooperation with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain.