Structural insights into the function of ZRANB3 in replication stress response

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations

PARP14 and PARP9/DTX3L regulate interferon-induced ADP-ribosylation

PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways

Updated protein domain annotation of the PARP protein family sheds new light on biological function

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.Work in I.A.’s group is funded by the WellcomeTrust (grant number 210634), BBSRC (BB/R007195/1), and Cancer ResearchUK (C35050/A22284). Work in D.A.’s group is funded by the Cancer ResearchUK Career Development Fellowship (grant number 16304). Work in the S.J.B.lab is supported by the Coun, which receives its core fundingfrom Cancer Research UK (FC0010048), the UK Medical Research Council(FC0010048), and the Wellcome Trust (FC0010048); a European Research Council (ERC) Advanced Investigator Grant (TelMetab); and Wellcome TrustSenior Investigator and Collaborative Grants. S.S.-B. was the recipient of an EMBO Long Term Fellowship (ALTF 707-2019) and a MSCA individual fellow-ship (grant 886577). Work in the J.R.C. group is funded by CRUK Career Devel-opment Fellowship (C52690/A19270) with infrastructural support from Well-come core award 090532/Z/09/ZS

Aminoacyl-tRNA Synthesis in Methanogenic Archaea

Aminoacyl-tRNA synthetases (AARSs) are essential for faithful translation of the genetic code and have long been studied intensively. Major discoveries explained basic principles of how amino acids are paired to their cognate tRNAs to ensure high fidelity of translation. However, advances in genomics instigated identification of novel enzymes and pathways to aminoacyl-tRNA synthesis. In that respect methanogenic Archaea are particularly prominent, most of which possess non-canonical routes to synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Additionally, some methanogenic seryl-tRNA synthetases are only marginally related to their homologues outside the archaeal kingdom, while other AARSs exhibit multiplicity of their genes (LysRS, SerRS, PheRS). Therefore, methanogens represent an exciting group of organisms regarding aminoacyl-tRNA synthesis, attesting to high degree of evolutionary diversity

Sinteza aminoacil-tRNA u metanogenih arhebakterija

Aminoacyl-tRNA synthetases (AARSs) are essential for faithful translation of the genetic code and have long been studied intensively. Major discoveries explained basic principles of how amino acids are paired to their cognate tRNAs to ensure high fidelity of translation. However, advances in genomics instigated identification of novel enzymes and pathways to aminoacyl-tRNA synthesis. In that respect methanogenic Archaea are particularly prominent, most of which possess non-canonical routes to synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Additionally, some methanogenic seryl-tRNA synthetases are only marginally related to their homologues outside the archaeal kingdom, while other AARSs exhibit multiplicity of their genes (LysRS, SerRS, PheRS). Therefore, methanogens represent an exciting group of organisms regarding aminoacyl-tRNA synthesis, attesting to high degree of evolutionary diversity.Aminoacil-tRNA-sintetaze su enzimi prijeko potrebni za vjernu translaciju genetskoga koda. Već su godinama cilj intenzivnih znanstvenih istraživanja, zahvaljujući kojima su razjašnjena osnovna načela sparivanja aminokiseline i pripadne molekule tRNA. Međutim, napredak u sekvenciranju novih genoma potaknuo je identifikaciju dotad nepoznatih enzima i puteva sinteze aminoacil-tRNA. Po tome su metanogene arhebakterije skupina osobitih svojstava jer nerijetko sadržavaju neuobičajene puteve sinteze asparaginil-tRNA, cisteinil-tRNA, glutaminil-tRNA te lizil-tRNA. Nadalje, pojedine metanogene seril-tRNAsintetaze pokazuju vrlo malu sličnost s vlastitim homolozima izvan arhejskog carstva, dok druge metanogene aminoacil-tRNA-sintetaze (lizil-tRNA-sintetaza, seril-tRNA-sintetaza i fenilalanil-tRNA-sintetaza) pokazuju višestrukost gena kojima su kodirane. Metanogene arhebakterije su zbog toga izrazito zanimljiva skupina organizama što se tiče sinteze aminoacil-tRNA te svjedoče o značajnoj evolucijskoj raznolikosti spomenutih biosintetskih puteva

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids

International audienceAlthough ubiquitylation had traditionally been considered limited to proteins, the disco v ery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study sho w ed that DTX2 E3 ligase efficiently ubiquitylates ADP r. Here, w e sho w that the ADP r ubiquit ylation activit y is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since str uct ural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might cat alyse ubiquit ylation of an ADPr moiety linked to nucleic acids. Indeed, w e sho w that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of re v ersible ubiquitylation of ADP-ribosylated nucleic acids

Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L

The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3′-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications

The interplay of TARG1 and PARG protects against genomic instability

The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ri-bosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine-and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability