Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish.

MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17-/- zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17-/- larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17-/- zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17-/- zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17-/- zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17-/- zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.

The Pol ß variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

DNA polymerase (Pol) ß is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol ß transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol ß (exon α Pol ß) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol ß variant in mouse embryonic fibroblasts. The exon α Pol ß variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol ß revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol ß variant is similar to that of wild-type Pol ß. These results indicate the exon α Pol ß variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol ß variant warrants further investigation.

AP endonuclease 1 (Apex1) influences brain development linking oxidative stress and DNA repair.

Brain and neurons are particularly sensitive to reactive oxygen species (ROS). Oxidative damage from ROS results in increased 8-oxoguanine in DNA followed by repair through the base excision repair (BER) pathway. We reported earlier that AP endonuclease 1 (Apex1) not only participates directly in BER but also regulates transcription factor Creb1. Here, we investigated how Apex1 affects brain to respond effectively to oxidative damage during zebrafish development. Loss of Apex1 resulted in increased ROS, 8-oxoguanine, and abasic sites as well as loss of Ogg1, which recognizes 8-oxoguanine and is required for its repair. Moreover, knock-down of Apex1 not only resulted in reduction of expression of several major proteins in the BER pathway (Polb and Ogg1), and it also resulted in maldistribution and loss of four key brain transcription factors (fezf2, otx2, egr2a, and pax2a), leading to abnormal brain development. These results were independent of p53 protein level. In contrast, exposure to exogenous H2O2 resulted in increased transcription and protein of Apex1 along with other BER components, as well as Creb1. Taken together, these results indicate that oxidative stress increased when the level of Apex1 was reduced, revealing a novel pathway of how Apex1 manages oxidative stress in developing brain.

Mechanistic toxicity of DEHP at environmentally relevant concentrations (ERCs) and ecological risk assessment in the Three Gorges Reservoir Area, China.

Di-(2-ethylhexyl) phthalate (DEHP) associated in vitro/vivo toxicity at current environmentally relevant concentration (ERC) with attendant ecological risks in the Three Gorges Reservoir Area (TGRA) is still elusive. Responding to this challenge, a novel integrated study based on analytical and biological assays was designed to elucidate the underlying mechanisms for toxicity of DEHP and its ecological risks at ERC. In this study, GC-MS analysis showed that the highest environmental concentration of DEHP in the TGRA surface water was nearly double that of WHO and USEPA standards. Both distribution and ecological risk decreased from the upper to middle and lower reaches of the TGRA. In vitro toxicity was assessed by cell viability and DNA damage assays: DEHP exposure at ERCs (100-800 µg/L) caused significant reduction in cell viability and elevated DNA damage. Further, DEHP exposure above 400 µg/L resulted in enhanced migration behavior of cancer cells. For in vivo toxicity assessment, short term acute exposure (7 d, 400 µg/L) apparently activated the PI3K-AKT-mTOR pathway, and chronic low-level exposure (3 months, 10-33 µg/L) suppressed the hypothalamus pituitary thyroid (HPT) axis pathway in zebrafish. In addition, acute low-level exposure (5 d, 33-400 µg/L) to DEHP increased aryl hydrocarbon receptor (AhR) activity in Tg(cyp1a:gfp) zebrafish in a concentration-dependent manner. In short, DEHP at ERC has extended potential to induce diverse in vitro and in vivo toxicity at concentrations that also cause impairment of biochemical function in aquatic species of the TGRA.

Deletion of mstna and mstnb impairs the immune system and affects growth performance in zebrafish.

Myostatin (Mstn) is a negative regulator of muscle development in vertebrates. Although its function in muscle growth has been well studied in mammals and fish, it remains unclear whether or how mstn functions in the immune system. In this study, mstna-/- and mstnb-/- homozygous zebrafish were firstly generated using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). Deletion of mstnb but not mstna enhanced growth performance. Although survival rates under normal conditions were slightly decreased in both strains, mortality after dexamethasone-induced stress was increased by ∼30%. Furthermore, transcriptional levels of several critical immune-related genes were decreased, and the ability to withstand exposure to pathogenic E. tarda was decreased, compared with that of controls. In mstnb-/- but not mstna-/- zebrafish, expression of NF-κB subunits and several pro-inflammatory cytokines failed to respond to E. tarda exposure except nfkb1, c-rel and tnfα. Taken together, these results indicate that mstnb but not mstna plays a key role in zebrafish muscle growth. While each paralogue contributes to the response to bacterial insult, mstnb affects the immune system through activation of the NF-κB pathway, and mstna is likely to act upstream of NF-κB at some as yet unidentified target.

A novel forward osmosis system in landfill leachate treatment for removing polycyclic aromatic hydrocarbons and for direct fertigation.

Landfill leachate (LL) is harmful to aquatic environment because it contains high concentrations of dissolved organic matter, inorganic components, heavy metals, and other xenobiotics. Thus, the remediation of LL is crucial for environmental conservation. Here, a potential application of the forward osmosis (FO) filtration process with ammonium bicarbonate (NH4HCO3) as a draw solution (DS) was investigated to remediate membrane bioreactor-treated LL (M-LL). After the leachate treatment, the toxicity and removal efficiencies of polycyclic aromatic hydrocarbons (PAHs) were evaluated using zebrafish and cultured human cells. The water recovery rate was improved using the current protocol up to 86.6% and 91.6% by both the pressure retarded osmosis (PRO) mode and the forward osmosis (FO) mode. Water flux increased with the increasing DS concentrations, but solution velocities decreased with the operation time. Toxicity tests revealed that the M-LL treated by NH4HCO3 had no toxic effect on zebrafish and human cells. Moreover, green fluorescent protein (GFP) expression in the transgenic zebrafish Tg(cyp1a:gfp) induced by PAHs was very weak compared to the effects induced by untreated M-LL. Since the diluted DS met local safety requirements of liquid fertilizer, it could be directly applied as the liquid fertilizer for fertigation. In conclusion, this novel FO system using NH4HCO3 as the DS provides a cheap and efficient protocol to effectively remove PAHs and other pollutants in LL, and the diluted DS can be directly applied to crops as a liquid fertilizer, indicating that this technique is effective and eco-friendly for the treatment of different types of LL.

Transcription Factors and DNA Repair Enzymes Compete for Damaged Promoter Sites.

Transcriptional regulation is a tightly regulated, vital process. The transcription factor cyclic AMP-response element-binding protein 1 (CREB1) controls ∼25% of the mammalian transcriptome by binding the CREB1 binding site consensus sequence (CRE) sequence (TGACGTCA). DNA lesions within CRE modulate CREB1 binding negatively and positively. Because appropriate DNA lesions also interact with base excision repair proteins, we investigated whether CREB1 and repair glycosylases compete with each other. We incubated 39-mer CRE-containing double-stranded oligonucleotides with recombinant CREB1 alone or with UNG2 or OGG1, followed by EMSA. The CpG islet within CRE was modified to contain a G/U or 8-oxoG (°G)/C mispair. OGG1 and CREB1 reversibly competed for CRE containing an °G/C pair. Also, OGG1 blocked CREB1 from dimerizing by 69%, even when total CREB1 binding was reduced only by 20-30%. In contrast, bound CREB1 completely prevented access to G/U-containing CRE by UNG2 and, therefore, to base excision repair, whereas UNG2 exposure prevented CREB1 binding. CREB1 dimerization was unaffected by UNG2 when CREB1 bound to CRE, but was greatly reduced by prior UNG2 exposure. To explore physiological relevance, we microinjected zebrafish embryos with the same oligonucleotides, as a sink for endogenous CREB1. As predicted, microinjection with unmodified or lesion-containing CRE, but not scrambled CRE or scrambled CRE with a G/U mispair, resulted in increased embryo death. However, only the G/U mispair in native CRE resulted in substantial developmental abnormalities, thus confirming the danger of unrepaired G/U mispairs in promoters. In summary, CREB1 and DNA glycosylases compete for damaged CRE in vitro and in vivo, thus blocking DNA repair and resulting in transcriptional misregulation leading to abnormal development.

DNA modifications repaired by base excision repair are epigenetic.

CREB controls ∼25% of the mammalian transcriptome. Small changes in binding to its consensus (CRE) sequence are likely to be amplified many fold in initiating transcription. Here we show that DNA lesions repaired by the base excision repair (BER) pathway modulate CREB binding to CRE. We generated Kd values by electrophoretic mobility shift assays using purified human CREB and a 39-mer double-stranded oligonucleotide containing modified or wild-type CRE. CRE contains two guanine residues per strand, one in a CpG islet. Alterations in CRE resulted in positive or negative changes in Kd over two orders of magnitude depending on location and modification. Cytosine methylation or oxidation of both guanines greatly diminished binding; a G/U mispair in the CpG context enhanced binding. Intermediates in the BER pathway at one G residue or the other resulted in reduced binding, depending on the specific location, while there was no change in binding when the single G residue outside of the CpG islet was oxidized. CREB recruits other partners after dimers form on DNA. Only UpG increased DNA.CREB dimer formation. Since oxidation is ongoing and conversion of cytosine to uracil occurs spontaneously or at specific times during differentiation and development, we propose that BER substrates are epigenetic and modulate transcription factor recognition/binding.

Zebrafish as a model system to study DNA damage and repair.

Zebrafish (Danio rerio) have become a popular vertebrate model to study embryological development, because of unique advantages not found in other model systems. Zebrafish share many gene functions with other vertebrates including humans, making zebrafish a useful system for studying cancer etiology. However, systematic studies of DNA damage and repair pathways using adult or embryonic zebrafish have not been extensively reported. The zebrafish genome contains nearly all the genes involved in different DNA repair pathways in eukaryotes, including direct reversal (DR), mismatch repair (MMR) nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR), non-homologous end joining (NHEJ) and translesion synthesis (TLS). It also includes the genes of the p53-mediated damage recognition pathway. Therefore, zebrafish provide an ideal model for gaining fundamental insights into mechanisms of DNA damage and repair, especially during embryological development. This review introduces recent work on different DNA damage and repair studies in zebrafish, with special emphasis on the role of BER in zebrafish early embryological development. AP endonuclease 1 (Apex1), a critical protein in the BER pathway, not only regulates BER but also controls cyclic AMP response binding protein (Creb1), which itself regulates ∼25% of eukaryotic coding sequences. In addition, Apex1 indirectly regulates levels of p53. As these findings also occur in murine B cells, they illustrate the usefulness of the zebrafish system in elucidating fundamental mechanisms.

Adenomatous polyposis coli interacts with flap endonuclease 1 to block its nuclear entry and function.

In previous studies, we found that adenomatous polyposis coli (APC) blocks the base excision repair (BER) pathway by interacting with 5'-flap endonuclease 1 (Fen1). In this study, we identify the molecular features that contribute to the formation and/or stabilization of the APC/Fen1 complex that determines the extent of BER inhibition, and the subsequent accumulation of DNA damage creates mutagenic lesions leading to transformation susceptibility. We show here that APC binds to the nuclear localization sequence of Fen1 (Lys(365)Lys(366)Lys(367)), which prevents entry of Fen1 into the nucleus and participation in Pol-ß-directed long-patch BER. We also show that levels of the APC/Fen1 complex are higher in breast tumors than in the surrounding normal tissues. These studies demonstrate a novel role for APC in the suppression of Fen1 activity in the BER pathway and a new biomarker profile to be explored to identify individuals who may be susceptible to the development of mammary and other tumors.

A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase beta.

DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex(+/-) mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes.

Conformational transitions in human AP endonuclease 1 and its active site mutant during abasic site repair.

AP endonuclease 1 (APE1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5' to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2'-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 x 10(4)-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis.

Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP endonuclease activity.

The base excision repair (BER) pathway recognizes and repairs most nonbulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages, and adults. Using a 45-mer double-stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER. Before 3 days postfertilization (dpf), aphidicolin-sensitive polymerases perform most nucleotide insertion. In fact, eggs and early stage embryos lack DNA polymerase-beta protein. After the eggs have hatched at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase-beta, becomes the primary polymerase. Previously, we showed that when the zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early stage embryos and adults are the absence of DNA polymerase-beta, leading to predominance of replicative polymerases, and the presence of backup Mg(2+)-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs fully when the embryos hatch from the chorionic membrane and encounter normal oxidative stress.

Enzymatic mechanism of human apurinic/apyrimidinic endonuclease against a THF AP site model substrate.

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 10(5) potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double-stranded DNA oligomers containing tetrahydrofuran (THF) with a 5'-phosphorothioate linkage as the abasic site substrate. Using H(2)(18)O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one-step associative phosphoryl transfer mechanism on a THF-containing substrate.

An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination.

Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2.

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease.

AP endonuclease (AP endo), a key enzyme in repair of abasic sites in DNA, makes a single nick 5' to the phosphodeoxyribose of an abasic site (AP-site). We recently proposed a novel mechanism, whereby the enzyme uses a key tyrosine (Tyr(171)) to directly attack the scissile phosphate of the AP-site. We showed that loss of the tyrosyl hydroxyl from Tyr(171) resulted in dramatic diminution in enzymatic efficiency. Here we extend the previous work to compare binding/recognition of AP endo to oligomeric DNA with and without an AP-site by wild type enzyme and several tyrosine mutants including Tyr(128), Tyr(171) and Tyr(269). We used single turnover and electrophoretic mobility shift assays. As expected, binding to DNA with an AP-site is more efficient than binding to DNA without one. Unlike catalytic cleavage by AP endo, which requires both hydroxyl and aromatic moieties of Tyr(171), the ability to bind DNA efficiently without an AP-site is independent of an aromatic moiety at position 171. However, the ability to discriminate efficiently between DNA with and without an AP-site requires tyrosine at position 171. Thus, AP endo requires a tyrosine at the active site for the properties that enable it to behave as an efficient, processive endonuclease.

DNA repair protein involved in heart and blood development.

Apurinic/apyrimidinic endonuclease 1, a key enzyme in repairing abasic sites in DNA, is an embryonic lethal in mice. We are examining its role in embryogenesis in zebra fish. Zebra fish contain two genomic copies (zfAPEX1a and zfAPEX1b) with identical coding sequences. zfAPEX1b lacks introns. Recombinant protein (ZAP1) is highly homologous with and has the same enzymatic properties as its human orthologue. ZAP1 is highly expressed throughout development. Embryos microinjected with morpholino oligonucleotide (MO) targeting the translation start site die at approximately the midblastula transition (MBT) without apoptosis. They are rescued with mRNA for human wild-type APEX1 but not for APEX1 encoding endonuclease-defective protein. Rescued embryos develop dysmorphic hearts, pericardial edema, few erythrocytes, small eyes, and abnormal notochords. Although the hearts in rescued embryos form defective loops ranging from no loop to one that is abnormally shaped, cardiac myosin (cmlc2) is present and contraction occurs. Embryos microinjected with MO targeting zfAPEX1a intron-exon junctions also pass the MBT with similar abnormalities. We conclude that AP endonuclease 1 is involved in both repairing DNA and regulating specific early stages of embryonic development.

Novel role of tyrosine in catalysis by human AP endonuclease 1.

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5' to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr(128) and Tyr(269), which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr(171) is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000-50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr(171) mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr(171) is the nucleophile that attacks the scissile phosphate.

A quantitative method for measuring protein phosphorylation.

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).