Genetic dissection of the biochemical activities of human DNA repair protein, APE1

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. Here we propose to identify and characterize critical amino acids of APE1 involved in either BER and/or NIR functions by using the alignment of the known three-dimensional (or tertiary) structures of Xth family AP endonucleases including the Methanothermobacter thermautotrophicus Mth212, Bacillus subtilis ExoA (1), E. coli Xth and human APE1 proteins (2)

Genetic dissection of the biochemical activities of human DNA repair protein, APE1

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. Here we propose to identify and characterize critical amino acids of APE1 involved in either BER and/or NIR functions by using the alignment of the known three-dimensional (or tertiary) structures of Xth family AP endonucleases including the Methanothermobacter thermautotrophicus Mth212, Bacillus subtilis ExoA (1), E. coli Xth and human APE1 proteins (2)

Multiple ciliary localization signals control INPP5E ciliary targeting

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Further-more, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoesMinisterio de Ciencia e Innovación PID2019-104941RB-I00 Raquel Martin-Morales, Pablo Barbeito Francesc R Garcia-Gonzalo. Ministerio de Ciencia e Innovación PID2020-114148RB-I00 Manuel Izquierdo. Instituto de Salud Carlos III CIBERER-ACCI-2020 Francesc R Garcia-Gonzalo. National Institutes of Health R01HD099784 Sarah C Goetz. American Heart Association 20POST35220046 Abhijit Deb Roy. Comunidad de Madrid PEJD-2016/BMD-2341 Belen Sierra-Rodero Ministerio de Ciencia e Innovación BES2016-077828 Raquel Martin-Morale

Completed Genomic Sequence ofBacillus thuringiensisHER1410 Reveals aCry-Containing Chromosome, Two Megaplasmids, and an Integrative Plasmidial Prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been completed, while the other draft genomes are incomplete, hindering full plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-read techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368 kb), a likely conjugative plasmid involved in virulence, and pLUSID2 (156 kb) potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome causes the disruption of a flagellar key component. Finally, phylogenetic analysis placed this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B. cereus s. s in agreement with the intermingled taxonomy of B. cereus sensu lato group.status: publishe

African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages.

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the Km and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3′→5′ exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage

Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway

Background: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to formation of etheno(ε)-bases including 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5’ to a damaged base in a DNA glycosylase-independent manner. Methodology/Principal Findings: Here, we characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. The activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has high affinity to DNA containing ε-bases but cleaves DNA duplex at an extremely slow rate. Consistent with this observation, the oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC50 values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates as expected DNA fragments containing 5’-terminal ε-base residue. Conclusions/Significance: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggest that NIR may act as a backup pathway to BER one to remove a large variety of genotoxic base lesions in human cells

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

ABSTRACTPrimary cilia are sensory membrane protrusions whose dysfunction causes diseases named ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, such as cilium stability, trafficking, signaling, or exovesicle release. Despite its key ciliary roles, how INPP5E accumulates in cilia remains poorly understood. Herein, we show that INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four ciliary localization signals (CLSs), the first two of which we newly discover: LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial redundancy between CLS1 and CLS4 explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 on the catalytic domain surface clarifies why CLS3 and CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations in INPP5E catalytic domain affect its ciliary targeting, and shed light on the mechanisms of action of each CLS. Thus, we find that CLS2 and CLS3 promote interaction with TULP3 and ARL13B, while downregulating CEP164 binding. On the other hand, CLS4 recruits PDE6D, RPGR and ARL13B, and cooperates with CLS1 in ATG16L1 binding. Lastly, we show INPP5E immune synapse targeting is CLS-independent. Altogether, we reveal unusual complexity in INPP5E ciliary targeting mechanisms, likely reflecting its multiple key roles in ciliary biology.</jats:p