Mouse NEIL1 protein is specific for excision of 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from oxidatively damaged DNA

A functional homologue of human DNA glycosylase NEIL1 (hNEIL1) in mouse has recently been cloned, isolated, characterized, and named mouse NEIL1 (mNEIL1). This enzyme exhibited specificity for excision of oxidatively modified pyrimidine bases such as thymine glycol, 5,6-dihydrouracil, and 5-hydroxypyrimidines, using oligonucleotides with a single base lesion incorporated at a specific site. It also acted upon AP sites; however, no significant excision of 8-hydroxyguanine was observed [Rosenquist, T. A., Zaika, E., Fernandes, A. S., Zharkov, D. O., Miller, H., and Grollman, A. P. (2003) DNA Repair 2, 581-591]. We investigated the substrate specificity and excision kinetics of mNEIL1 for excision of oxidatively modified bases from high-molecular weight DNA with multiple lesions, which were generated by exposure of DNA in aqueous solution to ionizing radiation. Among a large number of pyrimidine- and purine-derived lesions detected and quantified in DNA, only purine-derived lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine were significantly excised. This finding establishes that mNEIL1 and its functional homologue hNEIL1 possess common substrates, namely, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine. Measurement of excision kinetics showed that mNEIL1 possesses equal specificity for these two formamidopyrimidines. This enzyme also excised thymine-derived lesions thymine glycol and 5-hydroxy-5-methylhydantoin, albeit at a much lower rate. A comparison of the specificity and excision kinetics of mNEIL1 with other DNA glycosylases shows that this enzyme is as efficient as those DNA glycosylases, which specifically remove the formamidopyrimidines from DNA.     

Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.     

Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.     

8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein

We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantitative real-time PCR to measure the changes in gene expression caused by the presence of oxidised guanine residues generated by photosensitisation in the vector DNA. In wild-type cells, small numbers (one or three) of oxidised guanines did not affect gene expression at short times after transfections, whereas progressive reduction of the transgene expression was observed at later time points. Although Ogg1 has a major contribution to the repair of oxidised guanine bases, its absence did not have a strong effect on the gene expression. In contrast, the lack of functional Csb protein caused a pronounced inactivation of the damaged reporter gene. Most strikingly, an additional Ogg1 deficiency significantly attenuated this effect. The results indicate that the processing of oxidative guanine modifications by Ogg1 can mediate host cell inactivation rather than reactivation of the damaged genes and that this effect is strongly enhanced in the absence of Csb.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Formation of 8-hydroxyguanine residues in DNA treated with 4-hydroxyaminoquinoline 1-oxide and its related compounds in the presence of seryl-AMP

The mechanism whereby treatment of DNA with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP leads to the formation of 8-hydroxyguanine (8-OH-Gua) residues in DNA (Kohda et al., this journal, 139, 626, 1986) has been studied. In the survey of other N-arylhydroxylamines, only 4HAQO analogues which could bind to DNA produced 8-OH-Gua. The amount of 8-OH-Gua varied depending on the structure of 4HAQO analogues and that of DNA. The formation of 8-OH-Gua was not inhibited by active oxygen scavengers. Possible mechanisms are discussed.     

Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.     

Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana

It has long been known that animal heterotrimeric Gαβγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gβγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gβγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gβγ dimer directly and in a nucleotide-dependent manner. Although animal Gβγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gβγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gβγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals.     

Arabidopsis WD repeat domain55 Interacts with DNA damaged binding protein1 and is required for apical patterning in the embryo

CUL4-RING ubiquitin E3 ligases (CRL4s) were recently shown to exert their specificity through the binding of various substrate receptors, which bind the CUL4 interactor DNA damaged binding protein1 (DDB1) through a WDxR motif. In a segregation-based mutagenesis screen, we identified a WDxR motif-containing protein (WDR55) required for male and female gametogenesis and seed development. We demonstrate that WDR55 physically interacts with Arabidopsis thaliana DDB1A in planta, suggesting that WDR55 may be a novel substrate recruiter of CRL4 complexes. Examination of mutants revealed a failure in the fusion of the polar cells in embryo sac development, in addition to embryo and endosperm developmental arrest at various stages ranging from the zygote stage to the globular stage. wdr55-2 embryos suggest a defect in the transition to bilateral symmetry in the apical embryo domain, further supported by aberrant apical embryo localization of DORNROESCHEN, a direct target of the auxin response factor protein monopteros. Moreover, the auxin response pattern, as determined using the synthetic auxin-responsive reporter ProDR5:green fluorescent protein, was shifted in the basal embryo and suspensor but does not support a strong direct link to auxin response. Interestingly, the observed embryo and endosperm phenotype is reminiscent of CUL4 or DDB1A/B loss of function and thus may support a regulatory role of a putative CRL4(WDR55) E3 ligase complex.     

A diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) hydrolase from Arabidopsis thaliana that is activated preferentially by Mn2+ ions

Asymmetrical diadenosine 5',5'-P(1)P(4) tetraphosphate (Ap(4)A) hydrolases are key enzymes controlling the in vivo concentration of Ap(4)A--an important signaling molecule involved in regulation of DNA replication and repair, signaling in stress response and apoptosis. Sequence homologies indicate that the genome of the model plant Arabidopsis thaliana contains at least three open reading frames encoding presumptive Ap(4)A hydrolases: At1g30110, At3g10620, and At5g06340. In this work we present efficient overexpression and detailed biochemical characteristics of the AtNUDX25 protein encoded by the At1g30110 gene. Aided by the determination of the binding constants of Mn(Ap(4)A) and Mg(Ap(4)A) complexes using isothermal titration calorimetry (ITC) we show that AtNUDX25 preferentially hydrolyzes Ap(4)A in the form of a Mn(2+) complex.     

Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.     

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.     

Different roles of protein dynamics and ligand migration in non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana

The ligand rebinding kinetics after photolysis of the CO complexes of Arabidopsis thaliana hemoglobins AHb1 and AHb2 in solution show very different amplitudes in the geminate phase, reflecting different migration pathways of the photodissociated ligand in the system of internal cavities accessible from the heme. The dependence of the geminate phase on CO concentration, temperature, encapsulation in silica gels and presence of glycerol confirms a remarkable difference in the internal structure of the two proteins and a dramatically different role of protein dynamics in regulating the reactivity with CO. This finding strongly supports the idea that they have distinct physiological functions.     

Arabidopsis thaliana telomeric DNA-binding protein 1 is required for telomere length homeostasis and its Myb-extension domain stabilizes plant telomeric DNA binding

Telomeres are specific protein–DNA complexes that protect the ends of eukaryotic chromosomes from fusion and degradation and are maintained by a specialized mechanism exerted by telomerase and telomere-binding proteins (TBPs), which are evolutionarily conserved. AtTBP1 is an Arabidopsis thaliana protein that binds plant telomeric DNA in vitro. Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation. DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA. Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.     

Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair,somatic and meiotic recombination

Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.     

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two P_IB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu⁺-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.     

Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: in vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA

The discovery of 5-hydroxymethyl-cytosine (5hmC) in mammalian cells prompted us to look for this base in the DNA of Arabidopsis thaliana (thale cress), and to ask how well the Arabidopsis Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes this novel base. We found that the DNA of Arabidopsis' leaves and flowers contain low levels of 5hmC. We also cloned and expressed in Escherichia coli full-length VIM1 protein, the archetypal member of the five Arabidopsis VIM gene family. Using in vitro binding assays, we observed that full-length VIM1 binds preferentially to hemi-methylated DNA with a single modified 5mCpG site; this result is consistent with its known role in preserving DNA methylation in vivo following DNA replication. However, when 5hmC replaces one or both cytosine residues at a palindromic CpG site, VIM1 binds with approximately ≥10-fold lower affinity. These results suggest that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication.