Mutations defective in ribonucleotide reductase activity interfere with pollen plastid DNA degradation mediated by DPD1 exonuclease

Organellar DNAs in mitochondria and plastids are present in multiple copies and make up a substantial proportion of total cellular DNA despite their limited genetic capacity. We recently demonstrated that organellar DNA degradation occurs during pollen maturation, mediated by the Mg(2+) -dependent organelle exonuclease DPD1. To further understand organellar DNA degradation, we characterized a distinct mutant (dpd2). In contrast to the dpd1 mutant, which retains both plastid and mitochondrial DNAs, dpd2 showed specific accumulation of plastid DNAs. Multiple abnormalities in vegetative and reproductive tissues of dpd2 were also detected. DPD2 encodes the large subunit of ribonucleotide reductase, an enzyme that functions at the rate-limiting step of de novo nucleotide biosynthesis. We demonstrated that the defects in ribonucleotide reductase indirectly compromise the activity of DPD1 nuclease in plastids, thus supporting a different regulation of organellar DNA degradation in pollen. Several lines of evidence provided here reinforce our previous conclusion that the DPD1 exonuclease plays a central role in organellar DNA degradation, functioning in DNA salvage rather than maternal inheritance during pollen development.     

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.     

Gossypol mediated DNA degradation

Gossypol, a polyphenolic compound isolated from cotton plant was found to degrade pBR322 DNA in vitro in a reaction which required the presence of a metal ion, a reducing agent (2-mercaptoethanol) and oxygen as revealed after agarose gel electrophoresis. Fe3+ and Co2+ showed maximum degradation whereas addition of Ca2+ and Mg2+ prevented the gossypol mediated DNA damage. Gossypol caused degradation of rat liver DNA incubated in vitro even in the absence of added metal ions and 2-mercaptoethanol. Incubation of intact rat liver nuclei with gossypol revealed DNA degradation and nuclei isolated from rats treated with gossypol in vivo showed higher susceptibility to DNA fragmentation when incubated with gossypol in vitro than control nuclei. EcoR1 and AIuI digestion of DNA isolated from gossypol treated rats gave clear cut evidence for DNA degradation. These observations indicate that gossypol is genotoxic and considerable care has to be exercised in its use.     

DNA damage-induced MDMX degradation is mediated by MDM2

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.     

DNA end resection by CtIP and exonuclease 1 prevents genomic instability

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity.     

Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.     

Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.     

Geldanamycin-induced degradation of Chk1 is mediated by proteasome

Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.     

The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo

The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli. This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme. To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant. Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain. Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme.     

Construction of recombinant DNA by exonuclease recession.

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and site-directed mutagenesis.     

Ordered organelle degradation during starvation-induced autophagy

Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route. Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.     

Double-strand DNA break formation mediated by flap endonuclease-1

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.     

beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons

Alzheimer disease (AD) is a progressive, neurodegenerative disorder that leads to debilitating cognitive deficits. Although little is known about the early functional or ultrastructural changes associated with AD, it has been proposed that a stage of synaptic dysfunction might precede neurodegeneration in the development of this disease. Unfortunately, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently we have shown that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that plays a critical role in synaptic vesicle recycling, and hence, in the signaling properties of the synapse. We report here that this dynamin 1 degradation was the result of calpain activation induced by the sustained calcium influx mediated by N-methyl-D-aspartate receptors in hippocampal neurons. In addition, our results showed that soluble oligomeric Abeta, and not fibrillar Abeta, was responsible for this sustained calcium influx, calpain activation, and dynamin 1 degradation. Considering the importance of dynamin 1 to synaptic function, these data suggest that Abeta soluble oligomers might catalyze a stage of synaptic dysfunction that precedes synapse loss and neurodegeneration. These data also highlight the calpain system as a novel therapeutic target for early stage AD intervention.     

Synchronous digestion of SV40 DNA by exonuclease III.

We have established an optimal condition for the synchronous digestion of SV40 DNA with Escherichia coli exonuclease III. Electron microscopy and polyacrylamide gel electrophoresis were used to obtain accurate measurements on the lengths of DNA before and after exonuclease III digestion. Based on this finding, a new method for determining the sequence of long duplex DNA can be realized. It involves (a) the synchronous digestion of the DNA from the 3' ends with exonuclease III, followed by (b) repair synthesis with labeled nucleotides and DNA polymerase, and (c) sequence analysis of the repaired DNA.