Biomolecular condensates at sites of DNA damage: More than just a phase

Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates

MULTIDIMENSIONAL PEPTIDE/PROTEIN ANALYSIS AND IDENTIFICATION BY SEQUENCE DATABASE SEARCH USING MASS SPECTROMETRIC DATA

In order to generate proteomics data that are suitable to validate protein identification in complex mixtures using multidimensional liquid-chromatography-mass spectrometry approaches, we implemented an offline two-dimensional liquid chromatography method combining strong cation-exchange- and ion-pair reversed-phase chromatography followed by electrospray ionization tandem mass spectrormetry (ESI-MS/MS) for the analysis of a bovine serum albumin digest. The fragment ion spectra generated by ESI-MS/MS were subsequently analyzed via MASCOT database search. The obtained identification data were evaluated in terms of quality of protein/peptide identification by means of score values, reproducibility of identification in replicate measurements, distribution of tryptic peptides among different fractions, and overall number of unique identified proteins/peptides. Finally, we improved the trapping conditions in the second dimension by using a more hydrophobic amphiphile in the loading buffer. The improvement was demonstrated by comparison of the obtained identification data, such as number of identified peptides, cumulative mowse scores and reproducibility of identification

Iron regulation in the myxobacterium Myxococcus xanthus DK1622

The myxobacterium Myxococcus xanthus DK1622 is a reliable producer of different secondary metabolites with partially unknown bioactivities. In the present work the response of iron availibility were evaluated, concerning effects on growth, proteome profile and secondary metabolite production. The production of the siderophore myxochelin A was increased by the factor 81, myxochelin B by the factor 678. Unexpectedly, several other secondary metabolite production rates were found influenced, as e.g. myxochromids und cittilins. In proteome analysis, 1979 protein spots were detected in average, whereof 172 exhibited an iron-induced change in expression. A subsequent analysis by tandem mass spectrometry identified 169 of these spots as 131 individual proteins, some with up to 3 protein-phosphorylations. Furthermore, the functions of some, interesting proteins were investigated by knockout of the respective coding gene. At all, 12 single crossover mutants were generated and compared in iron-rich environment concerning effects on growth and rates of iron uptake or secondary metabolite production to the wild type strain. Typically, mutant strains show variations in all three parameters. An in-frame deletion mutant in one of the two fur genes (MXAN_6967) exhibited reduced growth and a decrease in iron uptake (ca. 49 % of the wild type). Additionally, production of all seven monitored secondary metabolites cannot be explained with the traditional Fur model, which suggests a new, unexpected regulation in M. xanthus.Das Myxobakterium Myxococcus xanthus DK1622 ist ein verlässlicher Produzent verschiedenster Sekundärmetabolite mit teilweise unbekannten biologische Aktivitäten. In der vorliegenden Arbeit wurde der Einfluss der Verfügbarkeit von Eisen auf Wachstum, Proteom-Muster und Sekundärmetabolit-Produktion untersucht. Die Produktion der Siderophore Myxochelin A wurde bei Eisenlimitierung um den Faktor 81 gesteigert, Myxochelin B um den Faktor 678. Unerwarteterweise wurden auch weitere Sekundärmetabolite-Produktionen stark beeinflusst, wie z.B. Myxochromid und Cittilin. In Proteomexperimenten konnten von durchschnittlich 1979 detektierten Proteinspots für 172 eine eiseninduzierte Konzentrationsveränderung gezeigt werden. Hiervon wurden 169 Spots mittels Tandem-Massenspektrometrie identifiziert als 131 individuelle Proteine mit bis zu 3 Phosphorylierungen. Des Weiteren wurde die Rolle von interessanten Proteinen durch Knockout entsprechender, codierender Genom-Bereiche untersucht. Insgesamt konnten 12 single-crossover Mutanten generiert werden, welche in eisenreicher Umgebung bezüglich Wachstum, Eisenaufnahme-Raten und Sekundärmetabolit-Produktionsraten mit dem Wildtyp-Stamm verglichen wurden, wobei die Mehrzahl Abweichungen vom Wildtyp in allen drei Parametern zeigten. Eine in-frame Deletionsmutante von einem der beiden fur-Gene (MXAN_6967) zeigte im Verglich mit dem Wildtyp reduziertes Wachstum. Die Verminderung der Eisenaufnahmeraten (49 % des Wildtyps) und Abnahme der Produktionsraten alle sieben Sekundärmetabolte kann mit dem traditionellen Fur-Model nicht erklärt werden, was eine neue, unerwartete Regulation bei M. xanthus nahe legt

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family member

The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

PARP inhibitors (PARPi) cause synthetic lethality in BRCA-deficient tumors. Whether specific vulnerabilities to PARPi exist beyond BRCA mutations and related defects in homology-directed repair (HDR) is not well understood. Here, we identify the ubiquitin E3 ligase TRIP12 as negative regulator of PARPi sensitivity. We show that TRIP12 controls steady-state PARP1 levels and limits PARPi-induced cytotoxic PARP1 trapping. Upon loss of TRIP12, elevated PARPi-induced PARP1 trapping causes increased DNA replication stress, DNA damage, cell cycle arrest, and cell death. Mechanistically, we demonstrate that TRIP12 binds PARP1 via a central PAR-binding WWE domain and, using its carboxy-terminal HECT domain, catalyzes polyubiquitylation of PARP1, triggering proteasomal degradation and preventing supra-physiological PARP1 accumulation. Further, in cohorts of breast and ovarian cancer patients, PARP1 abundance is negatively correlated with TRIP12 expression. We thus propose TRIP12 as regulator of PARP1 stability and PARPi-induced PARP trapping, with potential implications for PARPi sensitivity and resistance

An integrated magnet array for trapping and manipulation of magnetotactic bacteria in microfluidics

We present a novel system for localized magnetic manipulation of magnetotactic bacteria in microfluidic systems. Where other methods require small conductive tracks directly below the sample, the new system consists of an array of permanent magnets switchable by a drive current to either trap or guide bacteria. This allows for much higher magnetic fields at reduced power consumption. Both a theoretical analysis and experimental analysis are presented. The system is scalable and is suited for integration in microfluidics

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

BACKGROUND: The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1-69; however, this targeting sequence has not been experimentally examined or validated. RESULTS: Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin alpha3 and to a very weak extent with importin alpha1 and importin alpha5, the mutant PARP-2 (K36R) did not interact with importin alpha3, providing a molecular explanation why PARP-2 (K36R) is not targeted to the nucleus. CONCLUSION: Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions

Analysis of the PARP1, ADP-Ribosylation, and TRIP12 Triad With Markers of Patient Outcome in Human Breast Cancer

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently limit the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed the expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy

The iron-sulfur helicase DDX11 promotes the generation of single-stranded DNA for CHK1 activation

The iron-sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation