Structural and functional analysis of Rv3214 from Mycobacterium tuberculosis, a protein with conflicting functional annotations, leads to its characterization as a phosphatase

The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4'-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Angstroms resolution (R = 0.229; R(free) = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM alpha/beta structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4'-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.     

Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

Members of the archease superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing. Archease have therefore been predicted to play a modulator or chaperone role in selected steps of DNA or RNA metabolism, although the roles of archeases remain to be established experimentally. Here we report the function of one of these archeases from the hyperthermophile Pyrococcus abyssi. The corresponding gene (PAB1946) is located in a bicistronic operon immediately upstream from a second open reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease. In solution, the archease exists as a monomer, trimer, and hexamer. Only the oligomeric states bind the methyltransferase and prevent its aggregation, in addition to hindering dimerization of the methyltransferase-tRNA complex. This P. abyssi system possibly reflects the general function of archeases in preventing protein aggregation and modulating the function of their accompanying proteins.     

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.     

A systems biology approach reveals the role of a novel methyltransferase in response to chemical stress and lipid homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.     

Genetic and sequence analysis of an 8.7-kilobase Pseudomonas denitrificans fragment carrying eight genes involved in transformation of precorrin-2 to cobyrinic acid.

A 8.7-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and the genetic analysis revealed that this fragment carries eight different cob genes (cobF to cobM). Six of these genes have the characteristics of translationally coupled genes. cobI has been identified as S-adenosyl-L-methionine (SAM):precorrin-2 methyltransferase structural gene because the encoded protein has the same NH2 terminus and molecular weight as those of the purified enzyme. From protein homology with CobA and CobI, two SAM-dependent methyltransferases of the cobalamin pathway, it is proposed that cobF, cobJ, cobL, and cobM code for other methyltransferases involved in the cobalamin pathway. In addition, purified CobF protein has affinity for SAM, as expected for a SAM-dependent methyltransferase. Accumulation of cobalamin precursors in Agrobacterium tumefaciens mutants complemented by any of these eight genes suggest that, apart from cobI, whose function is identified, the products of all these genes are implicated in the conversion of precorrin-3 into cobyrinic acid.     

Solution structure of the conserved hypothetical protein Rv2302 from Mycobacterium tuberculosis

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel beta-sheet core with one C-terminal alpha-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the alpha-helix and beta-strands 3 and 4 hold the alpha-helix near the beta-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {(1)H}-(15)N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the (1)H-(15)N heteronuclear single quantum correlation spectrum suggests that this region, a loop between beta-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.     

Collectin structure: a review

Collectins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N-terminal region, a collagen like region, an alpha-helical coiled-coil, and the lectin domain. Only primary structure data are available for the N-terminal region, while the most important features of the collagen-like region can be derived from its homology with collagen. The structures of the alpha-helical coiled-coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP-D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP-D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP-D.     

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-l-methionine (AdoMet) has been determined at 1.98 A resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a beta-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively. Copyright 12001 Academic Press.     

The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.     

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.     

Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake

The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.     

Monomeric Nucleoprotein of Influenza A Virus

Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.     

Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: homooligomers of alpha-MSH

Homooligomers constructed with 4- and 6-amino acid fragments of melanocortin (alpha-MSH) bind with higher affinity and with apparent cooperativity to melanocortin receptor, compared to their constituent monomers. Individual ligands were tethered with various spacers of different length and rigidity and the influence of spacers on binding was studied. Binding assays were performed on cells transfected with the melanocortin receptor, hMC4R. There is a 5-7-fold decrease in the EC(50) with the addition of each subunit, going from monomer to trimer. The Hill coefficient increases from 0.76 for the monomer to 1.12 for the dimer and 1.35 for the trimer. These data show a general trend of increasing avidity with increasing number of ligands in oligomers.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

Crystal and solution structures of a superantigen from Yersinia pseudotuberculosis reveal a jelly-roll fold

Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.     

人PRMT5基因的生物信息学分析

PRMT5 (Protein arginine methyltransferase 5)已经广泛地作为组蛋白甲基转移酶修饰H4R3、H2AR3和H3R8,从而潜在地影响多个信号传导途径。本研究为了对人PRMT5基因进行生物信息学方面的深入分析,以PRMT5基因序列及编码蛋白序列为材料,通过生物信息学方法分析了PRMT5基因的DNA序列、启动子及CpG岛、RNA结构,及该蛋白的理化性质、亚细胞定位、信号肽与跨膜区域、互作蛋白、系统发育等。研究结果表明,人PRMT5在物种间的保守性相对较高,位于染色体14q11.2,大小为1 911 bp,潜在核心启动子在1 014~1 064 bp,没有预测到CpG岛。PRMT5基因编码637个氨基酸残基,分子量为10 157 Da,等电点为5.88,不稳定系数是44.33;该蛋白更可能定位于细胞质,无明显的信号肽及跨膜结构;主要的二级结构元件是α-螺旋结构和无规卷曲,包含一个SAM-dependent MTase功能结构域,同时有10个可能的互作蛋白;进化分析表明黑猩猩和猕猴与人PRMT5蛋白亲缘关系最近。     

Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike

Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer. The effects of pressure on two different tsf mutants, G244R and E196K, were explored. Pressure treatment of both G244R and E196K aggregates produced a folded trimer. E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein. In contrast, trimer formation from pressure-treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment. The center-of-mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates. Taken together, these results suggest that E196K has a primary defect in formation of the beta-helix during monomer collapse, while G244R is primarily an assembly defect.     

Direct structural evidence for a concerted allosteric transition in Escherichia coli aspartate transcarbamoylase

Regulation of protein function, often achieved by allosteric mechanisms, is central to normal physiology and cellular processes. Although numerous models have been proposed to account for the cooperative binding of ligands to allosteric proteins and enzymes, direct structural support has been lacking. Here, we used a combination of X-ray crystallography and small angle X-ray scattering in solution to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.     

Crystal structure of trimeric carbohydrate recognition and neck domains of surfactant protein A

Surfactant protein A (SP-A), one of four proteins associated with pulmonary surfactant, binds with high affinity to alveolar phospholipid membranes, positioning the protein at the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding, a characteristic of the collectin family, and specific interactions with lipid membrane components. The crystal structure of the trimeric carbohydrate recognition domain and neck domain of SP-A was solved to 2.1-A resolution with multiwavelength anomalous dispersion phasing from samarium. Two metal binding sites were identified, one in the highly conserved lectin site and the other 8.5 A away. The interdomain carbohydrate recognition domain-neck angle is significantly less in SP-A than in the homologous collectins, surfactant protein D, and mannose-binding protein. This conformational difference may endow the SP-A trimer with a more extensive hydrophobic surface capable of binding lipophilic membrane components. The appearance of this surface suggests a putative binding region for membrane-derived SP-A ligands such as phosphatidylcholine and lipid A, the endotoxic lipid component of bacterial lipopolysaccharide that mediates the potentially lethal effects of Gram-negative bacterial infection.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.