PAXX and its paralogues synergistically direct DNA polymerase λ activity in DNA repair

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5′ end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.This work was supported by the Medical Research Council (MRC) UK

NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy

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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

Structural studies of DNA complexes with minor groove-binding drugs

Targeting the minor groove of DNA with small molecules is an important recognition strategy in biology. A wide range of minor groove binding ligands (MGBLs) with good sequence discrimination ability are of interest as potential therapeutic agents in a variety of human diseases such as cancer, along with anti-bacterial and/or anti-parasitic activities. Whilst the detailed mechanism of action of some of these MGBLs is still unproven, they are known to be effective inhibitors of a number of minor and major groove binding protein-DNA interactions. This thesis reports on crystallographic studies to determine the molecular structure of MGBLs bound to DNA sequences, in order to better understand the details of their molecular recognition by DNA. Several interesting MGBLs differing in their structural features were crystallised with AT rich oligonucleotides for neutron and high-resolution X-ray data collection. Phases for the X-ray crystal structures were determined using molecular replacement, with diffraction data up to 1.2 A resolution. The crystal structure revealed the MGBLs bound in the central AATT or AAA TTT rich region of the minor groove of the DNA. The ligands form hydrogen bonds with the bases of the DNA at the floor of the minor groove directly or mediated via water molecules depending on the shape of the ligand. Several oligonucleotide-MGBL complexes were crystallised in the presence of deuterium oxide (heavy water) with the aim of studying the water network around the minor groove in the presence of ligand using neutron crystallography. In order to further our understanding of the biological mechanism of action ofMGBLs, biophysical studies were undertaken with the DNA major groove binding transcription factor, NF-KB. This transcription factor binds to the continuous guanine and cytosine bases of the major groove leaving the minor groove exposed to other molecules. Surface plasmon resonance (SPR) and small angle X-ray scattering (SAXS) studies were undertaken to study the effects on MGBLs on NF-KB - DNA binding. It was revealed that MGBLs had significant effect on the protein-DNA interactions which was further dependent on the shape of the MGBLs.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Basic research for clinical application of iPS cells in regenerative therapy for stroke

MacroD1 is a macrodomain containing protein that has mono-ADP-ribose hydrolase enzymatic activity toward several ADP-ribose adducts. Dysregulation of MacroD1 expression has been shown to be associated with the pathogenesis of several forms of cancer. To date, the physiological functions and sub-cellular localization of MacroD1 are unclear. Previous studies have described nuclear and cytosolic functions of MacroD1. However, in this study we show that endogenous MacroD1 protein is highly enriched within mitochondria. We also show that MacroD1 is highly expressed in human and mouse skeletal muscle. Furthermore, we show that MacroD1 can efficiently remove ADP-ribose from 5′ and 3′-phosphorylated double stranded DNA adducts in vitro. Overall, we have shown that MacroD1 is a mitochondrial protein with promiscuous enzymatic activity that can target the ester bonds of ADP-ribosylated phosphorylated double-stranded DNA ends. These findings have exciting implications for MacroD1 and ADP-ribosylation within the regulation of mitochondrial function and DNA-damage in vivo

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

International audienceAlphaFold2 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 pr o vides 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 pr o vides 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

Hierarchical Nanotube Self‐Assembly of DNA Minor Groove‐Binding Ligand DB921 via Alkali Halide Triggering

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