Internal Lys Residues Research Articles

PH-Driven Conformational Reorganization of Proteins: NMR Spectroscopy Study with Buried Lys Residues

The pH sensitivity of proteins is essential for biological energy transduction and for cellular and physiological homeostasis. The thermodynamic basis of pH effects in proteins is well understood but detailed mechanistic understanding of how changes in pH drive conformational reorganization in proteins is not. This was addressed systematically using NMR spectroscopy to characterize pH-dependent conformational changes coupled to the ionization of residues buried in the hydrophobic core of a protein. The study used 25 variants of staphylococcal nuclease with buried Lys residues. The pKa values of 19 of 25 of these internal Lys residues are depressed relative to the normal pKa of Lys in water, some by as much as 5 pH units. This shift in pKa ensures that the ionizable moiety stays neutral at pH values below 10 when it is buried, consistent with what is expected from the unfavorable transfer of a charge from water to a hydrophobic environment. In most cases, ionization of the buried Lys shifts the conformation of the protein into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of reorganization coupled to the ionization of buried Lys residues. The structural details of the conformational response were characterized with backbone chemical shift perturbation analysis, titrations of the buried Nζ atom, and in some cases even X-ray crystallography. At least in this protein, the anomalous pKa values of buried groups are governed by the propensity of the protein to reorganize. These results demonstrate that computational modeling of pH dependent processes in proteins will require prediction of alternative conformational states and accurate calculation of free energies; this remains a significant challenge even under the most favorable circumstances.

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15N-Detect NMR Spectroscopy.

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.

Conformational Exchange Coupled to Ionization of Lys Residues Buried in Hydrophobic Environments in Proteins

Ionizable amino acids buried in hydrophobic environments in proteins can titrate with anomalous pKa values. Despite the central role of these amino acids in energy transduction and catalysis, the determinants of their unusual properties are difficult to reproduce with computational approaches. Conformational reorganization of the protein backbone is a key determinant of pKa values. This reflects the ability of the protein to sample conformational microstates, which is particularly challenging to reproduce computationally. To characterize structural details and timescales of conformational reorganization coupled to ionization of buried groups, NMR spectroscopy was used to identify locations, measure timescales, and determine the extent of pH dependence of conformational exchange in the L25K and L125K variants of staphylococcal nuclease. Lys-25 or Lys-125 titrate with pKa values of 6.3 and 6.2, respectively. Using relaxation dispersion spectroscopy we observed that the β barrel of the L25K protein fluctuates between the native and an alternate state on the 0.4 to 7 ms timescale. The HN chemical shifts of the alternate state, assigned using ZZ-exchange spectroscopy, were less dispersed than those of the native state, consistent with unfolding of the β barrel. The equilibrium between these states is strongly pH dependent, with the partially unfolded state dominating at pH values at which the internal Lys residue is charged. In contrast, the L125K protein exhibited fluctuations near Lys-125 on the µs-ms timescale but in a faster regime than the L25K protein and without pH dependence. These results show the extent of coupling between conformational change and proton binding is highly dependent on local structural properties. Computational models attempting to reproduce the properties and pKa values of buried ionizable groups must be able to represent conformational exchange between native and subglobally unfolded states on ms timescales.

PKA Values of Buried Groups in Proteins are Sensitive to the Global Thermodynamic Stability

Ionizable groups buried in hydrophobic environments in proteins are essential for all forms of biological energy transduction. The molecular determinants of the pKa values of these internal groups are poorly understood. It is increasingly apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pKa values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pKa should be sensitive to the global thermodynamic stability (ΔG°) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pKa of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pKa of the internal Lys residues was found to be sensitive to the ΔG° of the protein confirming that the pKa values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pKa calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.

ChemInform Abstract: The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

AbstractReview: ca. 70 refs.

Structural Reorganization Triggered by the Ionization of Lys Residues buried in Hydrophobic Environments

A study of the structural consequences of ionization of amino acid side chains buried in the hydrophobic interior of proteins was performed by systematic introduction of lysine at 25 internal positions in a highly stable form of staphylococcal nuclease. Crystal structures for 12 of the variants at pH values where the Lys was expected to be uncharged revealed nearly identical backbones with the reference protein and ordered internal Lys side chains at the substitution site. The structures demonstrate that neutral Lys can be buried in the protein interior without the need for any specialized structural adaptations save for those required to stabilize the protein. Solvent-accessible surface area and depth of burial calculations indicated that all but one Lys were completely inaccessible to water. Seven Lys side chains packed into predominantly hydrophobic regions; five had Nζ atoms within 3.5 A of one or more polar groups or crystallographic water molecules. Depth of burial or microenvironment polarity as observed in the crystal structures did not correlate with measured shifts in pKa. 1H-15N HSQC NMR spectroscopy studies revealed global unfolding coupled to the ionization of two of the 25 Lys residues. Three others showed partial or local unfolding, five demonstrated localized changes in structure and dynamics, and 15 exhibited no detectable consequences. Backbone amide resonances were most similar between reference protein and the Lys-containing variant when the Lys side chains were buried shallowly or in a loop region. In the least stable variants the ionization of the internal Lys residues led to localized increase in conformational fluctuations or to conformational reorganization. These data will be useful to test the ability of structure-based pKa calculations to account for structural rearrangements or increased dynamics in response to ionization of internal groups.

The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

The conjugation of ubiquitin (Ub) to proteins is involved in the regulation of many processes. The modification serves as a recognition element in trans, in which downstream effectors bind to the modified protein and determine its fate and/or function. A polyUb chain that is linked through internal lysine (Lys)-48 of Ub and anchored to an internal Lys residue of the substrate has become the accepted "canonical" signal for proteasomal targeting and degradation. However, recent studies show that the signal is far more diverse and that chains based on other internal linkages, as well as linear or heterologous chains made of Ub and Ub-like proteins and even monoUb, are recognized by the proteasome. In addition, chains linked to residues other than internal Lys were described, all challenging the current paradigm.

Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.

Probing Affinity and Ubiquitin Linkage Selectivity of Ubiquitin-Binding Domains Using Mass Spectrometry

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.

Large shifts in pK a values of lysine residues buried inside a protein

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.

Strong Coulomb Interactions Between Internal and Surface Charges in Proteins

Internal ionizable groups in proteins are essential for many biological processes. The molecular determinants of their pKa values are poorly understood. To examine this problem, we measured previously the pKa values of Lys, Arg, Asp and Glu at 25 internal positions in staphylococcal nuclease. The pKa values are usually shifted substantially (as many as 6 pKa units) in the direction that favors the neutral state because dehydration experienced by the ionizable groups in their buried positions is not compensated by interactions with polar or charged groups. A subset of variants with internal Lys residues showed evidence of interaction of the internal Lys with surface carboxylic groups. Using NMR spectroscopy and crystallography we have shown that internal Lys residues can have significant Coulomb interactions with surface Asp and Glu. Upon ionization the internal Lys residue remains internal and the proteins remain fully folded. The interactions between the internal Lys and surface carboxylic groups are long range (6.3 Å or more between charged atoms) and are mostly through protein. Long-range pairwise interactions as high as 2 kcal/mol have been measured. In some cases strong effects are governed by the sum of many weaker long-range interactions that cannot be decomposed experimentally into pairwise contributions. Overall, there is strong evidence that surface carboxylic groups stabilize the charged form of many internal Lys residues. The experimentally measured Coulomb interaction energies between internal and surface charges constitute invaluable constraints for benchmarking of structure-based electrostatics calculations. They also suggest a strategy for modifying the pKa value of active site residues by the engineering of surface charges.

The action of fish peptide Orpotrin analogs on microcirculation

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N-terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp-desH(1) , Orp-Nle and Orp-Pro/Ala, respectively). Only Orp-desH(1) could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N-terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications.

Structural Consequences of the Ionization of Internal Lys Residues in a Protein

Internal ionizable residues in proteins play important functional roles in a variety of biological processes. The molecular determinants of their pKa values are poorly understood. Previously we measured the pKa of Lys, Asp, Glu and Arg at 25 internal groups in staphylococcal nuclease. 98 of these 100 variants are fully folded and native-like at pH 7. The pKa values of the majority of these groups are perturbed, some by as much as 5 pKa units, all in the direction that favors the neutral state. NMR spectroscopy was used to examine the structural and dynamic consequences of ionization of the internal lysine residues. In 9 of 10 crystal structures of Lys-containing variants the Lys side chain is completely buried, some in entirely hydrophobic microenvironments. In some cases the buried amino group makes contact with polar residues. The NMR experiments showed that in two variants the ionization of an internal Lys causes global unfolding. In five variants the ionization of the internal Lys triggers local structural changes and increased dynamics. The presence of conformational exchange in response to ionization appears to be correlated with the global stability of the variant proteins. Surprisingly, in the majority of cases, the changes in structure coupled to the ionization of the internal Lys residues are modest. These data demonstrate that proteins can tolerate internal ionizable residues, even those that exhibit large shifts in pKa values, and even in their charged states. The internal charged groups somehow manage to become solvated without disrupting the overall fold of the protein.

Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the approximately 70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express beta-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates.

Interaction of Fibrin(ogen) with Apolipoprotein(a): Further Characterization and Identification of a Novel Lysine-Dependent Apolipoprotein(a)-Binding Site within the γ Chain 287−411 Region

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.

Human Homologs of Ubc6p Ubiquitin-conjugating Enzyme and Phosphorylation of HsUbc6e in Response to Endoplasmic Reticulum Stress

Ubiquitin-conjugating enzyme Ubc6p is a tail-anchored protein that is localized to the cytoplasmic face of the endoplasmic reticulum (ER) membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. We have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response, a feature of many ER-associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress-related and dependent on signaling of the PRK-like ER kinase (PERK). The phosphorylation site was mapped to Ser-184, which resides within the uncharacterized region linking the highly conserved catalytic core and the C-terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization, or interaction with a partner ubiquitin-protein isopeptide ligase. Assays of hsUbc6e(S184D) and hsUbc6e(S184E), which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6p homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these Ub-conjugating enzymes during ERAD processes.

N-terminal ubiquitination: more protein substrates join in

The ubiquitin–proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an ε-NH 2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the α-NH 2 group of the N-terminal residue. An important biological question relates to the evolutionary requirement for an alternative mode of ubiquitination.

Degradation of the Id2 developmental regulator: targeting via N-terminal ubiquitination

Degradation of cellular proteins via the ubiquitin–proteasome system (UPS) involves: (i) generation of a substrate-anchored polyubiquitin degradation signal and (ii) destruction of the tagged protein by the 26S proteasome with release of free and reusable ubiquitin. For most substrates, it is believed that the first ubiquitin moiety is conjugated to a ε-NH 2 group of an internal Lys residue. Recent findings indicate that for several proteins, the first ubiquitin moiety is fused, in a linear manner, to the free α-NH 2 group of the protein. Here, we demonstrate that the inhibitor of differentiation (or inhibitor of DNA binding) 2, Id2, that downregulates gene expression in undifferentiated and self-renewing cells, is degraded by the UPS following ubiquitination at its N-terminal residue. Lysine-less (LL) Id2 is degraded efficiently by the proteasome following ubiquitination. Fusion of a Myc tag to the N-terminal but not to the C-terminal residue of Id2 stabilizes the protein. Furthermore, deletion of the first 15 N-terminal residues of Id2 stabilizes the protein, suggesting that this domain serves as a recognition element, possibly for the ubiquitin ligase, E3. The mechanisms and structural motives that govern Id2 stability may have important implications to the regulation of the protein during normal differentiation and malignant transformation.

Isolation and characterization of modified species of a mutated (Cys 125–Ala) recombinant human interleukin-2

During purification of recombinant and mutated interleukin-2 (rhIL-2A 125) by reversed-phase–high-performance liquid chromatography, more and less hydrophobic fractions named MHF and LHF, respectively are discarded due to the presence of some unidentified forms of rhIL-2Ala 125. Using slow and linear gradients of acetonitrile, these fractions were further purified by RP-HPLC, analyzed by automatic Edman degradation, digested with trypsin and analyzed by electrospray ionization mass spectrometry. In all fractions, partial processing of the N-terminal Met residue was observed. In the LHF the Met 104 was partially oxidized as sulfoxide. Combining the selective and reversible blocking of tryptic peptides and cation-exchange chromatography, two unexpected C-terminal peptides were selectively isolated. Automatic N-terminal sequencing showed that one of these corresponded to the C-terminal peptide of rhIL-2Ala 125 linked to another 11 amino acids (AANDENYALAA) and the other corresponded to the C-terminal peptide of a truncated rhIL-2Ala 125 without the C-terminal threonine residue and the extension of the 11 amino acids previously mentioned. MHF contained a mixture of four species of rhIL-2A 125 monoacetylated at the N-terminus and at the ε-amino groups of internal Lys residues: 8, 32 and 48. Cys 58 was found as free cysteine and also covalently linked to M r 69 and 77 molecules. Covalent dimers of rhIL-2A 125, linked through disulfide bridges between Cys 58 and Cys 105 of different monomers were also found.

Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA.

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.