Monitoring DNA polymerase ß mitochondrial localization and dynamics.

Mouse fibroblasts lacking (null) DNA polymerase ß (pol ß) were transfected with fluorescently tagged pol ß and stained with biomarkers to allow visualization within living cells by confocal microscopy. Transient transfection resulted in varying pol ß expression levels. Separating cells into three groups based on pol ß fluorescence intensity and morphological distribution, permitted analysis of the concentration dependence and spatial distribution of cytoplasmic pol ß. Colocalization between pol ß and mitochondria was pol ß concentration dependent. A decrease in overlap with nucleoids containing mitochondrial DNA (mtDNA) was observed at the highest pol ß intensity where pol ß exhibits a tubular appearance, suggesting the ability to load elevated levels of pol ß into mitochondria readily available for relocation to damaged mtDNA. The dynamics of pol ß and mitochondrial nucleoids were followed by confocal recording of time series images. Two populations of mitochondrial nucleoids were observed, with and without pol ß. Micro-irradiation, known to form DNA single-strand breaks, in a line across nucleus and cytoplasm of pol ß stably transfected cells enhanced apparent localization of pol ß with mitochondria in the perinuclear region of the cytoplasm near the nuclear membrane. Exposure of pol ß expressing cells to H2O2 resulted in a time-dependent increase in cytoplasmic pol ß observed by immunofluorescence analysis of fixed cells. Further screening revealed increased levels of colocalization of pol ß with a mitochondrial probe and an increase in oxidative DNA damage in the cytoplasm. ELISA quantification confirmed an increase of an oxidative mitochondrial base lesion, 7,8-dihydro-8-oxoguanine, after H2O2 treatment. Taken together, the results suggest that pol ß is recruited to mitochondria in response to oxidatively-induced mtDNA damage to participate in mtDNA repair.

SIRT1 regulates cardiomyocyte alignment during maturation.

Cardiomyocyte elongation and alignment, a critical step in cardiomyocyte maturation starting from the perinatal stage, is crucial for formation of the highly organized intra- and inter-cellular structures for spatially and temporally ordered contraction in adult cardiomyocytes. However, the mechanism(s) underlying the control of cardiomyocyte alignment remains elusive. Here, we report that SIRT1, the most conserved NAD+-dependent protein deacetylase highly expressed in perinatal heart, plays an important role in regulating cardiomyocyte remodeling during development. We observed that SIRT1 deficiency impairs the alignment of cardiomyocytes/myofibrils and disrupts normal beating patterns at late developmental stages in an in vitro differentiation system from human embryonic stem cells. Consistently, deletion of SIRT1 at a late developmental stage in mouse embryos induced the irregular distribution of cardiomyocytes and misalignment of myofibrils, and reduced the heart size. Mechanistically, the expression of several genes involved in chemotaxis, including those in the CXCL12/CXCR4 and CCL2/CCR2/CCR4 pathways, was dramatically blunted during maturation of SIRT1-deficient cardiomyocytes. Pharmacological inhibition of CCL2 signaling suppressed cardiomyocyte alignment. Our study identifies a regulatory factor that modulates cardiomyocyte alignment at the inter-cellular level during maturation.

Shining light on the response to repair intermediates in DNA of living cells.

Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase ß (pol ß). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol ß in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol ß.

Inositol Pyrophosphate Synthesis by Diphosphoinositol Pentakisphosphate Kinase-1 is Regulated by Phosphatidylinositol(4,5)bisphosphate.

5-diphosphoinositol tetrakisphosphate (5-InsP7) and bisdiphosphoinositol tetrakisphosphate (InsP8) are 'energetic' inositol pyrophosphate signaling molecules that regulate bioenergetic homeostasis. Inositol pyrophosphate levels are regulated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks); these are large modular proteins that host a kinase domain (which phosphorylates 5-InsP7 to InsP8), a phosphatase domain that catalyzes the reverse reaction, and a polyphosphoinositide-binding domain (PBD). Here, we describe new interactions between these three domains in the context of full-length human PPIP5K1. We determine that InsP7 kinase activity is dominant when PPIP5K1 is expressed in intact cells; in contrast, we found that InsP8 phosphatase activity prevails when the enzyme is isolated from its cellular environment. We approach a reconciliation of this disparity by showing that cellular InsP8 phosphatase activity is inhibited by C8-PtdIns(4,5)P2 (IC50 approx. 40 ìM). We recapitulate this phosphatase inhibition with natural PtdIns(4,5)P2 that was incorporated into large unilamellar vesicles. Additionally, PtdIns(4,5)P2 increases net InsP7 kinase activity 5-fold. We oftlinedemonstrate that PtdIns(4,5)P2 is not itself a phosphatase substrate; its inhibition of InsP8 phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the PBD. Finally, we discuss the significance of PtdIns(4,5)P2 as a novel regulator of PPIP5K1, in relation to compartmentalization of InsP7/InsP8 signaling in vivo.

XRCC1 phosphorylation affects aprataxin recruitment and DNA deadenylation activity.

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1-/- cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1-/- and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.

XRCC1-mediated repair of strand breaks independent of PNKP binding.

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1-/- mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1-/- cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent H2O2 due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.

DNA polymerase ß: A missing link of the base excision repair machinery in mammalian mitochondria.

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) ß. Pol ß was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol ß. Mitochondria from pol ß-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol ß-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol ß and thus pol ß plays a role in preserving mitochondrial genome stability.

Role of the oxidized form of XRCC1 in protection against extreme oxidative stress.

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase ß (pol ß) is critical for recruitment of pol ß to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol ß, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol ß binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol ß. Expression of reduced XRCC1 does not affect sensitivity to MMS or H2O2. In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to H2O2 compared with wild-type XRCC1-expressing cells. There is no indication of enhanced H2O2-generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following H2O2 exposure, suggesting BER deficiency of H2O2-induced damage in the C12A expressing cells.

PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains.

We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 µM and InsP6=7 µM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 µM and 1-InsP7=43 µM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.