Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling. © 2023 The Author(s

Toward definition of the structure of work: The development of a general-purpose job analysis instrument

The purpose of the current investigation was to develop and validate a general-purpose job analysis questionnaire capable of analyzing all types and levels of jobs, and to use this questionnaire as a means to investigate the underlying structure of work. The instrument was developed using existing items from the Job Element Inventory (Cornelius & Hakel, 1978) and managerial, supervisory, executive, and professional items which were based on dimensions reported in the literature. After completion of a pilot study, the questionnaire was administered to incumbents from a sample of jobs from various public and private sector organizations, resulting in the collection of 395 questionnaire responses. The job ratings were subjected to an exploratory factor analyses, and five overall and 28 divisional dimensions were interpreted. A policy-capturing approach was used to assess the validity of the questionnaire; R\sp2s ranged from.38 to.68. A confirmatory factor analysis investigated the following competing hypotheses of work structure. (1) The dimensionality of work resembles three dimensions: Working with People and Data, Physical Activities and Related Environmental Conditions, and Using Machines and Equipment. This structure is similar to Fine and Wiley's (1971) theory of work, which includes three factors: Data, People, and Things. (2) The dimensionality of work resembles six dimensions: Information Input, Mental Processes, Work Output, Relationships with Other Persons, Job Context, and Other Job Characteristics. These dimensions resemble information-processing theory and the a-priori divisions used by McCormick, Jeanneret, & Mecham (1977) to catagorize work. A covariance analysis confirmed the first two factors of the first hypothesis, but only three of the factors from the second hypothesis. Implications of these findings are discussed

RAD51AP1 Is an Essential Mediator of Alternative Lengthening of Telomeres

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RAD51AP1 Is an Essential Mediator of Alternative Lengthening of Telomeres

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RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA

International audienceAlternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA

Regulation of ALT-associated homology-directed repair by polyADP-ribosylation

International audienceThe synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers