Ryan Baxley

DNA lesions can stall replication, generating ssDNA gaps that require filling to maintain genome stability. The E3 ubiquitin ligase RFWD3/FANCW targets RPA and PCNA to regulate replication dynamics and damage tolerance, but its roles in these processes and influence on cell mutability remain incompletely understood. Here, we show that RFWD3 prevents the accumulation of ssDNA gaps in UV-irradiated cells through its E3 ligase activity and interaction with RPA. Mutational signature analysis implicates RFWD3 in the spontaneous mutability of human cells and reveals its involvement in 6-4 photoproducts tolerance. Accordingly, photorepair of 6-4 photoproducts, but not of cyclobutane pyrimidine dimers, alleviates gap accumulation in RFWD3-depleted cells. RFWD3 KO has limited impact on PCNA ubiquitylation, but its depletion sharply decreases UV-induced RPA ubiquitylation. Moreover, cells expressing an RPA mutant that cannot be ubiquitylated accumulate ssDNA gaps epistatically with RFWD3 loss. We propose that RFWD3-dependent RPA ubiquitylation regulates post-replicative ssDNA gaps to promote helix-distorting lesion tolerance.

RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the current literature describing the contribution of RAD18 to HR in mammalian systems has not reached a consensus. To investigate this, we examined three independent RAD18-null human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e., HCT116 mutants were hyper-recombinogenic (hyper-rec). Interestingly, these phenotypes were linked to RAD18’s role in PCNA K164 ubiquitination, as HCT116 PCNA^K164R/+ mutants were also hyper-rec, consistent with previous studies in rad18^−/− and pcna^K164R avian DT40 cells. Importantly, the knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. We propose that the hierarchy of post-replicative repair and HR, intrinsic to each cell type, dictates whether RAD18 is required for suppression of hyper-recombination and that this function is linked to PCNA K164 ubiquitination.

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18’s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e . HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNA ^K164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18 ^-/- or pcna ^K164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

In this study Brunner et al., set out to identify factors that promote efficient DNA replication in cells experiencing oncogene-induced replication stress (RS). This strategy identified the E3 ubiquitin ligase SKP1-Cullin1-F-box protein (SCF)-F-box and leucine rich repeat protein 12 (FBXL12). Interestingly, FBXL12 was only needed to prevent RS and DNA damage in cellular models of oncogene-induced RS, but not in cell lines with low intrinsic RS. Proteomic mass spectrometry identified FA complementation group D2 (FANCD2) and FANCI as possible FBXL12 targets. Using several in vivo and in vitro techniques, the authors show that checkpoint kinase 1 (CHK1) phosphorylation of the FANCD2 N-terminus was required for the FANCD2-FBXL12 interaction. Furthermore, this interaction was necessary for the FBXL12 dependent polyubiquitination and subsequent degradation of FANCD2. In the absence of FBXL12, FANCD2 remains chromatin bound and exacerbates RS. Notably, neither CHK1 phosphorylation nor FBXL12 polyubiquitination was related to FANCD2 mono-ubiquitination at lysine 561, which is associated with canonical FA-pathway activation and needed for stable FANCD2 chromatin association. Overall, this study provides novel mechanistic insights underlying FA-pathway regulation during the RS response and highlights the observation that variations in this response are directly related to the genetic context of each experimental model.

Zhang and colleagues utilized proteomics of isolated chromatin segments (PICh) to characterize the telomeric proteome during break-induced telomere synthesis (BITS). Following double-stranded break (DSB) induction, the human telomeric proteome was enriched for factors associated with the replication stress response. In particular, the authors describe how ubiquitinated proliferating cell nuclear antigen (PCNA) coordinates template switching to promote telomeric break-induced replication. This PCNA ubiquitination is dependent on the E3 ubiquitin ligase radiation sensitive 18 (RAD18) which is critical for the recruitment of the sensitive to nitrogen mustard 1 A (SNM1A) nuclease. SNM1A, but not nucleases associated with classical homologous recombination (HR), was the critical nuclease to resect telomeric DNA that promotes template switching and subsequent DNA polymerase delta subunit 3 (POLD3) dependent DNA synthesis. Overall, this study highlights the roles of ubiquitinated PCNA and SNM1A nuclease in promoting template switching-dependent lesion bypass during break-induced replication at telomeres.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA–protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 ( USP7) for survival in TP53 -null retinal pigment epithelial cells. TP53 ^−/– /RNF4 ^−/– /USP7 ^−/– triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

In this report, Mannherz & Agarwal used CRISPR-Cas9 genetic screening to identify genes that regulate human telomere length. In addition to well established factors such as telomerase and the Shelterin complex, the authors identified both positive and negative regulators of thymidine (dT) nucleotide metabolism. Unexpectedly, supplementation with dT for several weeks in culture, but not other deoxyribonucleosides (dA, dG, dC), increased telomere length by several kilobases. Supplementation of cells with dT caused telomere elongation without detectable changes in population growth or increased replication stress. Telomere lengthening via the regulation of cellular dT pools was promoted by thymidine kinase 1 (TK1) but not TK2, as well as thymidylate synthase (TYMS), and was negatively regulated by sterile alpha motif and HD domain-containing protein 1 (SAMHD1). This lengthening occurred in a telomerase-dependent manner, but unexpectedly, the observed increases in telomerase activity were independent of the role of dT as a telomerase substrate. Finally, dT supplementation effectively elongated telomeres in wildtype induced pluripotent stem cells (iPSCs) and iPSCs derived from patients with telomere maintenance disorders. These final observations suggest that dT supplementation may be useful clinically for the treatment of patients with genetic disorders related to telomere dysfunction.

In this study, Wu et al. investigated the contributions of the translesion synthesis (TLS) DNA polymerase zeta (Pol ζ) and the replicative DNA polymerase delta (Pol δ) to mitotic DNA synthesis (MiDAS). Using either an inducible degron system or siRNA knockdowns, the authors demonstrated that the POLD1, REV3 and REV1 subunits were each essential for MiDAS in human cancer cell lines. As predicted, these proteins colocalized with FANCD2 foci, a canonical marker of loci directed towards MiDAS following low dose aphidicolin (APH) treatment to induce replication stress. The DNA polymerase sliding clamp PCNA also colocalized with FANCD2 and was required for MiDAS. Interestingly, post-translational ubiquitination, but not sumoylation, of PCNA at lysine 164 (K164) was critical for MiDAS. Furthermore, this modification to promote MiDAS was catalyzed by the E3 ubiquitin ligase RAD18, but not CDT2. Overall, Wu et al. present data supporting a model where ubiquitinated PCNA first recruits TLS polymerases to initiate MiDAS. Next, TLS polymerases are swapped for the more processive replicative polymerase Pol δ. Finally, the authors found that oncogene activation, specifically overexpression of cyclin E, induced replication stress and triggered the MiDAS pathway. Based on these insights, the authors propose that targeting polymerases specific to MiDAS may be a unique vulnerability of cancer cells undergoing chronic DNA replication stress. In this context, it is worthwhile to note that previous studies had proposed the utility of REV1 inhibitors to block gap filling in cells with deregulated PrimPol activity {1-3}. Although this is mechanistically different from REV1 function during MiDAS, both pathways aid in suppressing under-replicated DNA.

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway,SLF2andSMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.

In this study, Lanna et al. investigated how T-lymphocytes (T-cells) maintain telomeres to delay senescence. They describe a mechanism whereby a subset of T-cells can directly acquire telomeric DNA from antigen-presenting cells (APCs) and incorporate this DNA to extend their telomeres up to several kilobases. Following immunological synapse with T-cells, APCs package trimmed telomeres into extracellular vesicles for transfer. First, APCs must degrade the shelterin complex to de-protect telomeric DNA. Next, telomeric-zinc-finger associated protein (TZAP) catalyzes cleavage of telomere sequences. Telomere-containing extracellular vesicles also included several proteins involved in homologous recombination, with radiation sensitive 51 (RAD51) shown to be critical for incorporating telomeric sequences in T-cells. Interestingly, telomere transfer was more effective than telomerase overexpression at promoting in vitro T-cell expansion. Finally, the authors demonstrated that this process could occur in mouse T-cells in vivo and was able to significantly promote long-term immune protection in experimental models. Overall, this study describes an unexpected mechanism for telomere extension in mammalian cells, and may be important for improving in vitro culturing of immune cells for therapeutic applications.