Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5′-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5′-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5′-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5′-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5′-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.     

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

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Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of “deviant” Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide array of metabolic functions (reviewed in Ref. 1). Research addressing the biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter vinelandii led to the discovery of an operon (iscA^nifnifUSVcysE1) involved in the biosynthesis of [Fe-S] clusters (reviewed in Ref. 2). Subsequent experiments led to the finding of two more systems involved in the de novo biosynthesis of [Fe-S] clusters, the isc and the suf systems (3, 4). Like Escherichia coli, the genome of Salmonella enterica serovar Typhimurium encodes for the isc and suf [Fe-S] cluster biosynthesis machinery.Recent studies have identified a number of additional or non-isc/-suf-encoded proteins that are involved in bacterial [Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY, an iron-binding protein believed to be involved in iron trafficking and iron delivery (5–7); YggX, an Fe²⁺-binding protein that protects the cell from oxidative stress (8, 9); ErpA, an alternate A-type [Fe-S] cluster scaffolding protein (10); NfuA, a proposed intermediate [Fe-S] delivery protein (11–13); YtfE, a protein proposed to be involved in [Fe-S] cluster repair (14, 15); and CsdA-CsdE, an alternative cysteine desulferase (16).Analysis of the metabolic network anchored to thiamine biosynthesis in S. enterica identified lesions in three non-isc or -suf loci that compromise Fe-S metabolism as follows: apbC, apbE, and rseC (17–21). This metabolic system was subsequently used to dissect a role for cyaY and gshA in [Fe-S] cluster metabolism (6, 22, 23). Of these, the apbC (mrp in E. coli) locus was identified as the predominant site of lesions that altered thiamine synthesis by disrupting [Fe-S] cluster metabolism (17, 18).ApbC is a member of the ParA subfamily of proteins that have a wide array of functions, including electron transfer (24), initiation of cell division (25), and DNA segregation (26, 27). Importantly, ATP hydrolysis is required for function of all well characterized members of this subfamily, and all members contain a “deviant” Walker A motif, which contains two lysine residues instead of one (GKXXXGK(S/T)) (28). ApbC has been shown to hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence alignments of the central portion of these proteins and bacterial ApbC are shown in Fig. 1. HCF101 was demonstrated to be involved in chloroplast [Fe-S] cluster metabolism (29, 30). The CFD1, Npb35, and huNbp35 (formally Nubp1) proteins were demonstrated to be involved in cytoplasmic [Fe-S] cluster metabolism (31, 32). Ind1 was demonstrated to be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme NADH:ubiquinone oxidoreductase (33). There is currently no report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of ParA family of proteins. Protein alignments were assembled using the Clustal_W method in the Lasergene® software and show only the central portion of the proteins, which have the highest sequence conservation. The three boxed areas highlight the Walker A box, conserved Ser residue, and CXXC motif. Proteins listed are as follows: ApbC (S. enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35 (S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35 (formally Nubp1) (Homo sapiens), and Ind1 (Candida albicans).Biochemical analysis of ApbC indicated that it could bind and transfer [Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate isomerase (34). Additional genetic studies indicated that ApbC has a degree of functional redundancy with IscU, a known [Fe-S] cluster scaffolding protein (35, 36).In this study we investigate the correlation between the biochemical properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and [Fe-S] cluster transfer rates) and the in vivo function of this protein. This is the first detailed kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of deviant Walker A proteins and the first functional analysis of a member of the ever expanding family of ApbC/Nbp35 proteins. Data presented indicate that noncomplementing variants have distinct biochemical properties that place them in three distinct classes.     

Sites of Intra- and Intermolecular Cross-linking of the N-terminal Extension of Troponin I in Human Cardiac Whole Troponin Complex

Our previous studies (Howarth, J. W., Meller, J., Solaro, R. J., Trewhella, J., and Rosevear, P. R. (2007) J. Mol. Biol. 373, 706–722) of the unique N-terminal region of human cardiac troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional cross-linker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intra- and intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca²⁺-dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled troponin. Ca²⁺ regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca²⁺, but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca²⁺. This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra- and intermolecular sites.The human cardiac variant of troponin I (hcTnI)² has structural and functional specializations that are related to its critical role in control of cardiac dynamics. These specializations include variations in amino acids that are significant factors in the response of the heart to: adrenergic stimulation (1), sarcomere length (2, 3), and pH (4, 5). An especially significant region of specialization is a unique N-terminal extension of 30–32 amino acids, which contains serial serines at positions 23/24 that are substrates for kinases that control cardiac dynamics (6–8). Despite its significance in control of cardiac function, molecular mechanisms of how the N-terminal human cardiac troponin I (N-hcTnI) region controls sarcomeric and cardiac function remain poorly understood. There is evidence that upon phosphorylation the interaction between the N-hcTnI and the N-lobe of N-hcTnC is weakened (9, 10). The structure of the N-hcTnI was missing in the crystal structure of cardiac troponin (11). However, we recently reported (12) the structure of the N-terminal peptide using NMR. Docking of this structure into the core troponin structure indicated the potential for a previously unappreciated intramolecular interactions of the N terminus with the regions at or near the highly basic inhibitory peptide region of cardiac troponin (12, 13). This interaction appeared plausible not only on the basis of the structure of hcTnI, but also on the basis of the preponderance of basic amino acids in the inhibitory peptide and the presence of acidic residues in the N terminus.In experiments reported here, we tested the hypothesis that the unique N-terminal region of hcTnI engages in both intra- and intermolecular interactions. We introduced Cys residues into the N-hcTnI at positions 5 and 19 and labeled the Cys residue with the hetero-bifunctional cross-linker, BP-MAL, which upon UV irradiation cross-links to residues within ∼10 Å of the modified Cys (14). We analyzed the cross-linked peptides by Edman sequencing and mass spectrometry to determine specific sites of interaction. The intramolecular sites of interaction were Met-154 and Met-155 in the hcTnI switch peptide for labeled positions 19 and 5, respectively. The intermolecular cross-linking sites on N-hcTnC were 47 and 80 for hcTnI labeled at either position 5 or 19. Measurement of Ca²⁺-dependent ATPase rate in reconstituted preparations indicated that allosteric effects of the different specific intramolecular cross-links (position Met-154 versus Met-155) to different hydrophobic positions on the switch peptide may affect hcTnC interaction with the switch peptide.     

Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage

During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3′→5′ exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg²⁺ and Ca²⁺ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3′→5′ exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.     

hNOA1 Interacts with Complex I and DAP3 and Regulates Mitochondrial Respiration and Apoptosis

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O₂ consumption ∼20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as γ-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and dynamics (fission and fusion) are closely intertwined. Apoptosis and changes in metabolism are associated with morphological changes in mitochondria (1, 2). Conversely, when mitochondrial morphology is altered either by mutations or altered expression of mitochondrial fission or fusion proteins such as the dynamin like large G proteins Drp1 and Opa1, the cell''s susceptibility to apoptotic agents (3) or ability to generate ATP (4, 5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), and mitochondria play central roles in both pathways (6). The apoptotic pathways involve a growing list of mitochondria-associated proteins, such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located either on the outer mitochondrial membrane (OMM)³ or in the intermembrane space (IMS) (7). Recently, proteins of the mitochondrial matrix such as DAP3, have also been shown to be involved in apoptosis (8). DAP3 has been reported to be involved in both γ-interferon- (9) and tumor necrosis factor-α-induced (10) apoptosis as well as staurosporine-induced mitochondrial fragmentation (11), but the detailed mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of mitochondria in respiration and generation of ATP. The electron transport chain in the inner mitochondrial membrane (IMM) contains four major enzyme complexes (Complexes I, II, III, and IV) that are involved in transferring electrons from NADH (Complex I-linked) or FADH2 (Complex II-linked) to O₂ and in pumping protons out of the matrix to create an electrochemical proton gradient, which is harnessed by ATP synthase to make ATP (12).Despite the accumulating evidence showing intercommunication between mitochondrial metabolism, apoptosis, and dynamics, how these processes are coordinated remains to be elucidated. In this study we characterize hNOA1, the human homologue of Arabidopsis thaliana nitric oxide-associated protein, 1 (AtNOA1) (13). hNOA1 is a large G protein closely related to dynamin that is associated with the IMM. Perturbation of hNOA1 affects mitochondrial morphology, Complex I-linked O₂ consumption, and the cell''s susceptibility to apoptotic stimuli, possibly through interactions with proteins such as Complex I and DAP3.     

Interaction of Replication Protein A and Flap Endonuclease 1 with DNA Duplexes Containing a Nick or a Flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase (Pol) interaction with DNA. Using Pol, a photoreactive dTTP analog was added to the 3" end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5" end of a nick or a flap. Photoaffinity labeling of Pol substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol during long-patch base excision repair.     

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772–19780).The human pyruvate dehydrogenase complex (PDC)³ resides in mitochondria and catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA and reducing equivalents (NADH), serving as a link between glycolysis and the Krebs cycle (1–3). The PDC is a large (∼9.5 MDa) catalytic machine comprising multiple protein components. The three catalytic components are pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), with E3 being a common component between different α-keto acid dehydrogenase complexes. The two regulatory enzymes in the PDC are the isoforms of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.The PDC is organized around a structural core, which includes the C-terminal domains of E2p and a noncatalytic component that specifically binds E3, i.e. the E3-binding protein (E3BP). To this E2p/E3BP core, multiple copies of the other PDC components are tethered through noncovalent interactions. Each E2p subunit contains two consecutive N-terminal lipoic acid-bearing domains (LBDs), termed L1 and L2, followed by the E1p-binding domain (E1pBD) and the C-terminal inner-core/catalytic domain, with these independent domains connected by unstructured linkers. Similarly, each E3BP subunit consists of a single N-terminal LBD (referred to as L3), the E3-binding domain (E3BD), and the noncatalytic inner core domain. Together, the inner core domains of E2p and E3BP assemble to form the dodecahedral 60-meric E2p/E3BP core. The role of the E1pBD and E3BD domains is to tether E1p and E3, respectively, to the periphery of the E2p/E3BP core. It is presumed that the LBDs (L1, L2, and L3) shuttle between the active sites of the three catalytic components of the PDC during the oxidative decarboxylation cycle (4). The eukaryotic PDC is unique among α-keto acid dehydrogenase complexes in its requirement for E3BP; prokaryotic PDCs employ the single subunit-binding domain to secure either E1p or E3 to the complex (5).Using a “divide-and-conquer” approach, a wealth of structural information on the PDC has been accumulated recently. High-resolution crystal structures are available for the human E1p (6–8) and E3 components (9). A model for the human E2p has been constructed based on an 8.8-Å electron density map available from cryo-electron microscopy (10). Additionally, solution and crystal structures of the L1 and L2 domains of E2p have been determined (11–13), and the high-resolution crystal structures of the E3BD (14, 15), pyruvate dehydrogenase kinase isoforms 1–4 (12, 16–18), and pyruvate dehydrogenase phosphatase isoform 1 (19) are known. Therefore, atomic models are available for almost all components and domains of the mammalian PDC.With the successes of the above structural approach, attention has turned to the overall structure of the PDC. There are two outstanding questions as follows. What are the subunit and overall catalytic component stoichiometries? What are the positions and orientations of the components in this large catalytic machine? Yu et al. (10) recently determined the cryo-EM structure of a PDC core comprising only human E2p subunits. Like yeast E2p, human E2p adopts a dodecahedral structure composed of 60 E2p proteins; each face of the dodecahedron has a large gap. Although this structure is highly informative, the composition of this core deviates substantially from that of the native PDC, because no E3BP subunits are present in the core structure. Based on the similar structure of the dodecahedral yeast PDC, a hypothesis was formed that, in human PDC, 12 copies of E3BP bind in the 12 gaps, which is termed the “60/12” model (20). Biophysical studies on complexes of E2p and E3BP later negated the 60/12 model; Hiromasa et al. (21) therefore posited an alternative, the “48/12” model, in which the dodecahedral core includes 48 E2p subunits and 12 E3BP proteins. A further source of conjecture is how many E1p and E3 components bind to the periphery of the PDC. If one binding domain binds to one peripheral catalytic component, a maximally occupied 60/12 PDC would harbor 60 E1p heterotetramers and 12 E3 dimers (or 48 E1ps and 12 E3s in the 48/12 model). The notion of such 1:1 binding is supported by the preponderance of available biophysical evidence. Specifically, two crystal structures, site-directed mutagenesis, and calorimetric measurements describe a 1:1 interaction between E3BD and E3 (14, 15). Also, although no structures are available for the human E1p-E1pBD complex, a crystal structure of the homologs of these proteins from Bacillus stearothermophilus also demonstrates a 1:1 interaction between the E1pBD of E2p and the E1p heterotetramer (22). In addition, ITC experiments performed on the bacterial E1p and the cognate subunit-binding domain indicate a 1:1 association (23). At variance with the above observations, a different subunit stoichiometry has been proposed by Smolle et al. (24, 25). Their evidence suggests that two binding domains bind for every peripheral component; such an arrangement potentially yields a PDC with half as many peripheral components bound.This study was undertaken to ascertain the subunit and component stoichiometries of the human PDC, particularly with regard to interactions between the E3BD and the E3 dimer. We show that quantification of bands on an SDS-polyacrylamide gel of a PDC reconstituted at saturating E1p and E3 concentrations supports neither the 60/12 nor the 48/12 model. Instead, a “40/20” model is proposed, and subsequent ITC and analytical ultracentrifugation (AUC) data corroborate this new model. In addition, results from electrophoretic mobility shift assays, ITC, and AUC presented here uniformly show a 1:1 interaction between E3BD and the E3 dimer as well as between E1pBD and the E1p heterotetramer. The implications of this 1:1 binding stoichiometry for the macromolecular assembly of the PDC are discussed.     

UVI31+ Is a DNA Endonuclease That Dynamically Localizes to Chloroplast Pyrenoids in C. reinhardtii

UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.