The DUF328 family member YaaA is a DNA-binding protein with a novel fold

Janani Prahlad; Yifeng Yuan; Jiusheng Lin; Chou-Wei Chang; Dirk Iwata-Reuyl; Yilun Liu; Valérie de Crécy-Lagard; Mark A Wilson

doi:10.1074/jbc.RA120.015055

. 2020 Aug 12;295(41):14236–14247. doi: 10.1074/jbc.RA120.015055

The DUF328 family member YaaA is a DNA-binding protein with a novel fold

Janani Prahlad ^1,^‡, Yifeng Yuan ^2,^‡, Jiusheng Lin ¹, Chou-Wei Chang ⁴, Dirk Iwata-Reuyl ³, Yilun Liu ⁴, Valérie de Crécy-Lagard ^2,^5,^*, Mark A Wilson ^1,^*

PMCID: PMC7549036 PMID: 32796037

Abstract

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200–300 nm. YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

Keywords: DNA binding, comparative genomics, X-ray crystallography, bacterial stress response, UPF0246, DNA-binding protein, oxidative stress, bioinformatics, stress response

Bacteria confront diverse environmental stresses and mount defensive responses to ensure survival and growth. The accumulation of reactive oxygen species is a common stressor, causing cellular oxidative stress and eliciting a robust and multifaceted response in bacteria. In Escherichia coli, one of the primary sensors for oxidative stress is the OxyR transcription factor, which regulates the expression of ∼20–40 genes in response to oxidation of its regulatory cysteine residues (1, 2). Hydrogen peroxide is the dominant OxyR effector and therefore most of the genes controlled by OxyR are involved in the peroxide stress response (3). Although many of these downstream peroxide-responsive proteins have been extensively characterized in bacteria, some are surprisingly poorly understood.

One peroxide-responsive protein whose function is unclear is E. coli YaaA (YaaA_Ec, locus b0006), a member of the DUF328/UPF0246 family. YaaA_Ec is up-regulated in response to increased H₂O₂ levels (1), and it decreases intracellular Fe²⁺ levels in E. coli by an unknown mechanism (4). Fe²⁺ is cytotoxic in the presence of hydrogen peroxide because of the production of highly reactive hydroxyl radicals via Fenton chemistry (5). Therefore, the tight control of Fe²⁺ is important during peroxide stress and helps explain why YaaA_Ec is under the control of the OxyR regulon.

Gene expression array analysis showed that the transcription level of yaaA in E. coli is reduced in stationary phase and in anaerobic conditions and increased during recovery in LB broth from a stationary phase inoculum (6). This observation suggests that YaaA is important for the exponential phase of aerobic growth. In addition, the transcription levels of yaaA, yaaJ (a putative transport protein next to yaaA), and ten genes (fhuA, fhuF, fiu, cirA, entC, entB, exbD, fecI, fepB, and fepD) that are responsible for iron acquisition were considerably lower in exponential phase growth of an rpoS mutant compared with WT E. coli MG1655 (7), indicating that RpoS regulates YaaA in E. coli. The connection between iron, peroxide, and YaaA was bolstered by the recent report that a yaaA mutant of Klebsiella pneumonia has impaired survival in the presence of H₂O₂ in iron-replete culture conditions, whereas there is no difference in survival of WT and yaaA mutant K. pneumonia in oxidative stress under low-iron conditions (8).

Bioinformatic analyses of the DUF328 family have indicated a link between these proteins and nucleic acid metabolism, particularly to the metabolism of the 7-deazaguanosine–modified nucleosides (9). A DUF328 domain is found in DpdC proteins involved in the formation of 2′-deoxy-7-amido-7-deazaguanosine (dADG) from 2′-deoxy-7-cyano-7-deazaguanosine (dPreQ₀) in vivo (10). The 7-deazaguanosine derivatives dADG and dPreQ₀ are recently discovered DNA modifications encoded by the dpd cluster found in a diverse set of bacteria. Of the 11 genes found in the Salmonella serovar Montevideo dpd cluster (dpdA–dpdK), dpdC probably encodes an enzyme that hydrolyzes dPreQ₀ to dADG (10). E. coli K12 MG1655 has the queuosine-tRNA pathway but no dpd genes, suggesting that YaaA_Ec is likely to have a distinct function.

A further cellular connection between YaaA_Ec and DNA repair was reported by Liu et al. (4), who observed enhanced mutation rates in ΔyaaA E. coli and severe growth defects for ΔyaaA recA56 and ΔyaaA polA1 double mutant strains that had intact oxidative stress response enzymes when grown in an aerobic atmosphere. RecA is a ssDNA-binding protein that plays an important role in DNA recombination and repair (11). The recA56 mutant is defective in ATP-regulated formation of nucleoprotein filaments and can partially impair recombination by WT RecA (12). PolA (DNA polymerase I) is an E. coli DNA polymerase that is involved primarily in DNA repair; the polA1 mutation results in increased mutagenesis and diminished base mismatch repair (13, 14). The ΔyaaA recA56 and ΔyaaA polA1 double mutants displayed growth defects in aerobic growth conditions that otherwise required a severely impaired oxidative stress response to be observed in the ΔyaaA single mutant E. coli (4). In aggregate, these results suggest a functional link between YaaA_Ec, oxidative stress, and DNA repair. Despite a growing body of data about the role of YaaA and other DUF328 proteins in bacterial oxidative stress defense, little is known about any DUF328 protein at the molecular level, including their structures or functions.

Here, we provide a direct molecular connection between the DUF328 family, specifically YaaA_Ec, with DNA maintenance and the oxidative stress response in bacteria. Bioinformatic analysis shows a strong association between DUF328 proteins and genes involved in DNA recombination and oxidative stress response. We find that YaaA_Ec directly binds to DNA in vitro, and the 1.65-Å resolution X-ray crystal structure of YaaA_Ec shows that the protein possesses a new fold with a positively charged cleft and a helix-hairpin-helix (HhH) DNA-binding motif found in other proteins that bind DNA in a nonsequence-specific manner. As the first member of the DUF328 family to be structurally characterized, YaaA_Ec establishes a new protein fold family that we propose be named the cantaloupe fold. Our results show that the DUF328 family comprises DNA-binding proteins that play important roles in DNA maintenance during oxidative stress, assigning a molecular function to this family of proteins.

Results

Comparative genomic analyses show that DUF328 genes are widespread and are fused or physically clustered with genes involved in oxidative stress and DNA repair

YaaA_Ec is a member of the DUF328 (IPR005583/H2O2_YaaD/PF03883) family. Analysis of the taxonomic distribution of members of this family shows that it is widespread in bacteria (Fig. 1), with homologs found in ∼45.0% (10,565/23,458) of the reference organisms in the Genome Taxonomy Database (GTDB) (15). Phyla that harbor the most DUF328 family members are Campylobacterota (∼87.4%, 222/254), Actinobacteriota (∼83.9%, 2617/3118), Proteobacteria (∼59.1%, 4512/7630), and Bacteroidota (∼55.3%, 1573/2843), where the values in the parenthesis indicate the fraction of total reference organisms that contain DUF328 members. In contrast, DUF328 members are much less widespread in archaea, with homologs found in only ∼1.3% (31/2,392) of the reference organisms in the GTDB (Fig. S1).

Figure 1. — **Phylogenetic distribution of DUF328 proteins and representative genomes in bacteria.** A, results of an AnnoTree query using Pfam family PF03883 (DUF328) in bacteria with resolution at the genus level. Branches are highlighted in *blue* for phyla that harbor members of the DUF328 family. The trees were generated in AnnoTree with the e-value cutoff of 0.00001 and adapted for visualization (RRID:SCR_018980). B, representative genome neighborhood diagrams of PF03883 (DUF328) family (*red arrows*). YaaJ: PF01235 (sodium:alanine symporter family), QueF: PF14489 (QueF-like protein), 2OG: PF13640 (2-oxoglutarate-Fe(II) oxygenase superfamily), DEAD: PF00270 (DEAD/DEAH box helicase), helicase: PF00271 (helicase conserved C-terminal domain), RecQ-like: PF16124 (RecQ zinc-binding), adh: PF00106 (short-chain dehydrogenase), AhpC: PF00578 (alkyl hydroperoxide reductase subunit C/ thiol-specific antioxidant family), Nif: PF01784 (NIF3 (NGG1p interacting factor 3)), zf: PF02591 (C4-type zinc ribbon domain).

Genes with related functions often cluster together in bacterial genomes. To capture gene neighborhood associations of the DUF328 family, we performed a sequence similarity network (SSN) analysis combined with genome neighborhood network (GNN) analysis (16, 17). Using a stringent alignment score threshold of 90, YaaA_Ec (UniProt ID P0A8I3) is partitioned into the largest cluster (cluster 1) that contains no sequence with an associated gene ontology term (Fig. S2), indicating that YaaA_Ec is not closely associated with any sequence of annotated function.

The GNN analysis of genes encoding proteins from the major DUF328/PF03883 SSN clusters shows extensive clustering with genes involved in oxidative stress response (Figs. S3–S7). Of the nine extracted neighborhoods, four contain genes encoding alkyl hydroperoxide reductase/thiol-specific antioxidant proteins of the AhpC family (PF00578). Three contain genes encoding for Ruberythrin, a nonheme iron-containing metalloprotein involved in oxidative stress tolerance in anaerobic bacteria (PF02915). Two contain genes encoding oxidative dealkylation repair proteins of the AlkB family (PF13640) and members of the DUF3501 family (PF12007), previously linked to oxidative stress (18). No other functional area is as highly represented among the DUF328 GNN proteins, even though we see quite a few RNA metabolism and translation proteins (PF00270, PF04073, PF00587, PF00849, PF10150, PF14489, and PF14819).

GNN analysis links members of the DUF328 family to oxidative stress, and analysis of Rosetta stone–type protein domain fusions (19) indicates an association between DUF328 proteins and DNA maintenance and repair. Several DUF328 proteins are fused to domains from the GIY-YIG endonuclease superfamily (IPR000305) (Fig. 2). Nucleases of the GIY-YIG family are involved in DNA repair and recombination, transfer of mobile genetic elements, and restriction of incoming foreign DNA (20 –22). The GIY-YIG proteins are so-named because they contain a domain of typically ∼100 amino acids with two short motifs, “GIY” and “YIG”, in their N-terminal regions. Additionally, DUF328 domains are found fused to multiple recombinase domains, a Zn²⁺-β ribbon domain, a PadR helix-turn-helix motif, and an AlbA_2 DNA-binding domain (Fig. 2), underscoring the connection between DUF328 and DNA binding and repair. The gene fusion and neighborhood analyses corroborate the function of DUF328 proteins in oxidative stress first reported for the YaaA_Ec (4) but add a link to DNA repair that was not obvious from previous studies.

Figure 2. — **Schematics of examples of PF03883/IPR005583/DUF328 fusion proteins.** Representatives were retrieved from pfam and Conserved Domain Architecture Retrieval Tool. H2O2_YaaD (DUF328): PF03883; GIY-YIG: PF01541; SpoVK: COG0464; Ser_Rec: Ser_Recombinase superfamily, cd00338; Rec: recombinase, pfam07508; Zn: recombinase zinc β ribbon domain, pfam13408; AlbA_2: putative DNA-binding domain, PF04326; HTH: helix-turn-helix domains. aa, amino acids.

DUF328 genes show strong cofitness with the RecFOR pathway for DNA repair

A measure of the joint contribution of two or more genes to organism survival is given by the cofitness, which is the Pearson correlation coefficient of their contributions to organismal fitness under different physiological conditions. Cofitness analysis derived from an extensive Tn-Seq analysis of 32 organisms in dozens of conditions (23) further supports an association between DUF328 genes, DNA recombination, and hydrogen peroxide detoxification. In the case of E. coli, the top cofitness associations are with peptidoglycan synthesis and cell division genes (alr, ftsN and amiA) with scores between 0.45 and 0.48, and then with a few DNA repair genes such as recG and uvrD (scores of 0.44 and 0.43, respectively). However, none of these scores were extremely high. The highest cofitness scores (>0.75) were found in other species, indicating that DUF328 genes are likely to function in the same pathway with the DNA replication and repair protein coding gene recF in Shewanella sp. ANA-3, with catalase/peroxidase coding genes in Acidovorax sp. GW101-3H11, and with unknown genes in Pseudomonas syringae pv. syringae B728a ΔmexB and Dechlorosoma suillum PS (Table 1). The strong conserved cofitness (cofitness >0.6 and ortholog cofitness >0.6) indicates functional relationships of DUF328 genes with genes coding DNA repair and recombination proteins, such as recA and recN (Table 1). DUF328 genes from at least seven bacteria show strong conserved cofitness (ortholog cofitness >0.6 in Table 1 and Table S1) with components of the RecFOR pathway that initiates recombination-mediated DNA repair by processing ssDNA gaps and loading RecA onto the recombinogenic ends (24 –26). The highest ortholog cofitness scores (>0.75) include genes encoding the DNA replication and repair protein recF in Caulobacter crescentus and Pseudomonas fluorescens FW300-N2E2 (Table S1). Several other genes in the recFOR pathway (recA, recJ, recN, recO, and recR) also display high cofitness with DUF328 genes, pointing to a strong joint contribution to organism survival (Table 1 and Table S1). The recurrent cofitness association with exonuclease I gene (scbB) further corroborates the association between DUF328 proteins and ssDNA involved in recombination. Prokaryotic exonuclease I possesses a 3′–5′ single-stranded DNA exonuclease activity that is stimulated by ssDNA-binding protein and plays an important role in DNA repair (27). Also recurring in the DUF328 cofitness analysis is catalase, which defends against H₂O₂ stress by converting H₂O₂ to H₂O and O₂, reinforcing the connection between DUF328 proteins and peroxide stress.

Table 1.

Genes that show strong and conserved cofitness with DUF328 genes

Data extracted from the Fitness Browser (http://fit.genomics.lbl.gov/cgi-bin/myFrontPage.cgi)

Organism	YaaA homolog	Cofitness hit	Description	Ortholog cofitness	cofitness
Shewanella loihica PV-4	Shew_1093	Shew_1611	sbcB, exonuclease I	0.66	0.63
Shewanella sp. ANA-3	Shewana3_3143	Shewana3_1126	recA, recombinase A	0.63	0.72
		Shewana3_1104	recombination and repair protein	0.62	0.67
		Shewana3_2572	sbcB, exonuclease I	0.66	0.66
		Shewana3_0011	recF, recombination protein F (RefSeq)	0.57	0.75
Shewanella oneidensis MR-1	SO3540	SO1328	transcriptional regulator, LysR family	0.68	0.65
		SO3430	recA protein	0.72	0.63
Pseudomonas syringae pv. syringae B728a ΔmexB	Psyr_1064	Psyr_2850Psyr_3174	hypothetical proteinuroporphyrinogen-III C-methyltransferase/precorrin-2 dehydrogenase	nono	0.80.78
Dechlorosoma suillum PS	Dsui_2322	Dsui_2345	hypothetical protein	no	0.88
		Dsui_3354	adenine specific DNA methylase Mod	no	0.88
Caulobacter crescentus NA1000	CCNA_03496	CCNA_02062	DNA repair protein recN	0.67	0.62
Acidovorax sp. GW101-3H11	Ac3H11_1593	Ac3H11_784	hydrogen peroxide-inducible genes activator	0.65	0.68
		Ac3H11_2454	exodeoxyribonuclease I (EC 3.1.11.1)	0.66	0.66
		Ac3H11_452	polyphosphate kinase (EC 2.7.4.1)	0.49	0.8
		Ac3H11_135	catalase (EC 1.11.1.6)/peroxidase (EC 1.11.1.7)	no	0.79

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Recombinant YaaA_Ec binds DNA with nanomolar affinity

Consistent with strong bioinformatic evidence connecting DUF328 family proteins to DNA maintenance and repair, YaaA_Ec is associated with a large amount of nucleic acid during purification of the recombinant protein from E. coli. We determined that the associated nucleic acid was DNA based on its selective degradation by DNaseI. Ultimately, nearly all of the DNA was removed from YaaA_Ec by hydroxyapatite chromatography (see “Experimental procedures”), but strong anion exchange chromatography with a Q resin could not separate YaaA_Ec from the DNA. The persistence of DNA in association with YaaA_Ec suggests direct and tight binding.

YaaA_Ec binding to DNA was measured using EMSA with a variety of defined DNA structures. YaaA_Ec binds to double-stranded, bulge-containing, and Holliday junction DNA with comparable dissociation constants (K_D) of ∼200–300 nm (Fig. 3). YaaA_Ec also binds ssDNA, although with lower apparent affinity (Fig. 3A). Unlike many other DNA binding proteins, YaaA_Ec shows no strong preference for specific DNA structures. Multiple shifted DNA bands are observed at higher concentrations of YaaA_Ec in the EMSA, suggesting that these DNA constructs are capable of binding multiple copies of YaaA_Ec (Fig. 3, A–C). The fraction of the DNA duplex with a 12-nt bubble bound by YaaA_Ec was well-fitted by a cooperative binding model, giving a K_D = 265 nm and a Hill coefficient of ∼3.1 (Fig. 3D). All the EMSAs show evidence of positive cooperativity in YaaA_Ec binding, and several distinct bands are seen at higher concentrations of YaaA_Ec. Either multiple YaaA_Ec molecules can bind a single DNA molecule directly, or one YaaA_Ec binds to the DNA fragment and additional YaaA_Ec molecules interact with the bound YaaA_Ec in the YaaA_Ec-DNA complex. Our data do not discriminate between these two possibilities, although the Hill coefficient of 3.1 suggests a cooperative YaaA_Ec recruitment mechanism.

Figure 3. — **YaaA_Ec binds to diverse DNA constructs with nanomolar affinity.** *A–C*, EMSA of recombinant YaaA_Ec with various DNA constructs illustrated *below each panel*. The concentration of YaaA_Ec is shown at the *top of each panel* and the fraction of the total DNA signal that is shifted from the free position is quantified beneath each lane. D, the binding of YaaA_Ec to the 12 nt bubble duplex DNA from EMSA is fitted to a single-site, positively cooperative binding model. Data were measured in triplicate and verified for multiple preparations of YaaA_Ec. The Hill coefficient was fitted as 3.1.

YaaA_Ec role is PreQ₀-independent

DUF328 domains are present in DpdC proteins that are predicted dPreQ₀ nitrile hydratases (10), and DUF328 genes physically cluster with queF genes encoding PreQ₀ reductase (Fig. 1B). A parsimonious prediction is that YaaA_Ec may be involved in the recognition and repair of PreQ₀ that has been incorporated into DNA by mistake. Indeed, tRNA guanine transglycosylase (TGT), normally involved in the synthesis of queuosine in tRNA, can also insert the PreQ₀ derivative PreQ₁ in DNA in vitro if the thymine base is replaced with uracil (28). The presence of uracil in DNA does occur and is usually corrected by a specific repair machinery (29). However, the addition of exogenous PreQ₀ did not affect growth of the ΔyaaA mutant in LB (Fig. S8). In addition, the growth defect caused by the deletion of yaaA in the Hpx⁻ background (4) seemed to be improved by the addition of PreQ₀ and exacerbated by the deletion of the queD gene involved in the PreQ₀ synthesis pathway. Even if the difficulty of working with the Hpx⁻ strain makes this last result within the margin of error (Fig. S9), it does not fit with a role of YaaA in repairing potential misincorporations of PreQ₀ in DNA. Additional studies in which uracil levels are increased and PreQ₀ levels are measured in DNA would be needed to totally rule out this hypothesis, however.

YaaA_Ec possesses a new fold

We determined the X-ray crystal structure of YaaA_Ec to 1.65-Å resolution using single-wavelength anomalous diffraction (SAD) phasing of the selenomethionine (SeMet)-substituted protein. YaaA_Ec is a single-domain protein possessing a new fold comprising 12 α-helices and 14 β-strands with a core defined by a three-stranded parallel β-sheet (Fig. 4, A and B). Several of the secondary structural elements are short (e.g. αD, αG, αI, αJ, and β2, β3, β4, β8, β9) and may be differently classified by various secondary structure–detection algorithms. Overall, YaaA_Ec is wedge-shaped with an apical cleft, resembling a slice of cantaloupe (Fig. S10). Helices αB and αC compose a HhH DNA-binding motif (see below) that is positioned opposite a β-strand motif comprising β11–14. The β-strand motif has an unusual abundance of solvent-exposed aromatic amino acids and several lysine residues. The cleft region that lies between the HhH and β-strand motifs is ∼20 Å wide and is rich in basic residues, resulting in a highly positive electrostatic potential as calculated by the Adaptive Poisson-Boltzmann Solver (APBS) (30) (Fig. 4C). Residues in the cleft region display an approximate dyad symmetry (Fig. 4D) and are among the most highly conserved residues in the DUF328 family, including a ²⁰⁹KKARG²¹³motif that binds a chloride ion from the crystallization buffer. The positive electrostatic potential of the cleft, the rough dyad symmetry of several conserved residues in the region, and a width that matches the diameter of B-form DNA make this cleft a plausible contact surface for DNA.

Figure 4. — **Three-dimensional structure of YaaA_Ec defines a new fold.** A and B, two views of the ribbon diagram of YaaA_Ec with α-helices lettered and β-strands numbered. The views in (A) and (B) are related by the 90° rotation indicated by the *arrow*. YaaA_Ec defines a new protein fold class. C, the electrostatic potential surface for YaaA_Ec looking down into the apical cleft as calculated by APBS (see “Experimental procedures”). Electrostatic potential is colored from *blue* (+10 kT/e) to *red* (−10 kT/e), where T is 298 K. D, the same view into the apical cleft as (C) with key residues labeled. The *green* and *gold* residues compose two groups related by an approximate dyad axis that passes between residue pairs Glu¹³⁰, Tyr¹⁷⁵ and Lys⁹, Lys²⁰⁹.

YaaA_Ec has multiple extended stretches of polypeptides adopting nonstandard structures from residues 6–22, 67–78, and 123–135, interrupted by a short β-strand from residues 10–12. These distinctive and atypical polypeptide structures define a broad area on the exterior of YaaA_Ec that includes part of the cleft and also penetrate into the core of the protein (Fig. 5A). These regions are not loops as typically defined because they are not confined to the surface of the protein and meander through the core of the protein structure. Unlike loops, these atypically structured residues make extensive contacts with neighboring residues and are well-ordered judging from both the quality of the electron density (Fig. S11) and their low refined atomic displacement parameters (ADPs). The majority of the peptide atoms in these nonstandard secondary structural regions make hydrogen bonds with solvent or nearby amino acid sidechains rather than other peptides, again differentiating them from peptide groups in standard secondary structural elements such as α-helices and β-strands. Among these unusual contacts, a chain of Tyr-mediated hydrogen bonds extends across the conserved core of the region. This Tyr-rich hydrogen bond network includes a highly conserved SGXYG motif (¹¹²SGLYG¹¹⁶ in YaaA_Ec) that is located at a sharp turn between β-strands in the core of YaaA_Ec (Fig. 5B) and makes extended contacts with residues 67–72 and 124–131. The high degree of conservation (Fig. S12) and clear structural importance of the SGXYG motif indicates that the surrounding unusually structured regions are likely conserved features of the DUF328 family.

Figure 5. — **YaaA_Ec possesses long stretches of nonstandard structures extending from the core to the periphery of the protein.** A, a ribbon diagram of YaaA_Ec with the conserved SGXYG motif in *yellow* and the surrounding stretches of peptides adopting structures that are neither α-helix or β-strand in *magenta*. B, a close-up view of this region, where the *dotted lines* show a network of hydrogen bonds that mediate interactions between the conserved SGXYG motif and the surrounding residues. Tyrosine residues are highly represented in this region.

YaaA contains a HhH DNA-binding motif

Although YaaA_Ec possesses a new fold, the Phyre2 fold recognition server (31) identifies an HhH motif from residues 35–66 that is found in several DNA-binding proteins (Fig. 6A). HhH motifs bind DNA in Hef-domain RuvA domain 2-like proteins (PDB 2AQ₀, 1X2I, and 2BGW) (32 –34), excinuclease abc subunit c (PDB 1KFT) (35), DNA/RNA-binding 3-helical bundle, GerE-like (LuxR/UhpA family of transcriptional regulators) (PDB 1FSE) (36), DNA excision repair protein XPF-ERCC1 (PDB 6SXB) (37), and mitochondrial transcription elongation factor 2 (PDB 5OL9) (38), among others. In the structures that include bound DNA (6SXB and 2BGW), the HhH motif directly contacts the minor groove of DNA, suggesting that this is a possible DNA-binding mode for YaaA_Ec as well (Fig. 6B). A preference for binding the minor groove is consistent with the lack of sequence-specific DNA binding by HhH domains (39). The putative DNA-binding regions in the apical cleft of YaaA_Ec, including parts of the HhH motif, show the highest sequence conservation in the DUF328 family and strongly support the functional significance of these regions (Fig. S13).

Figure 6. — **YaaA_Ec has a HhH DNA-binding motif.** A, a ribbon diagram of YaaA_Ec with the region that is structurally conserved in other DNA-binding proteins colored *magenta*. The classical HhH motif is defined by αB and αC (*labeled*). B, a superposition of YaaA_Ec (*gray*, *blue*) with the HhH motif (*yellow*) and bound DNA from XPF-ERCC1 endonuclease (PDB 6SXB). This illustrates one potential DNA-binding mode for YaaA_Ec and other DUF328 proteins.

Discussion

In this study we show that DUF328 proteins, including the E. coli representative YaaA_Ec, are DNA-binding proteins involved in the oxidative stress response. Prior work from the Imlay group (4) showed that YaaA_Ec is important for regulating ferrous iron (Fe²⁺) levels in oxidatively stressed E. coli. Fe²⁺ is dangerous in the presence of H₂O₂ because Fenton chemistry can generate the highly reactive hydroxyl radical, which is indiscriminately destructive (40). The hydroxyl radical is thought to result in bacterial death predominantly through DNA damage (41). Although our results do not explain how YaaA_Ec regulates Fe²⁺ levels, they demonstrate that a key molecular activity of YaaA_Ec is DNA binding, indicating that it plays an important role in DNA maintenance and repair under oxidative stress conditions. This is consistent with prior observations that yaaA deletion resulted in a mutator phenotype and a filamentous E. coli cell morphology (4), which is a common cellular manifestation of extensive DNA damage in bacteria (42). Moreover, the multiple and strong comparative genomic connections between YaaA_Ec homologs and DNA repair suggest that this function is likely conserved throughout the DUF328 family.

Cofitness analysis reveals a strong connection between DUF328 proteins and the RecFOR pathway of single-stranded gap DNA repair. Although this indicates a connection between DUF328 proteins and DNA repair, it does not require that YaaA_Ec operate in the RecFOR pathway. Cofitness indicates that DUF328 and RecFOR genes make joint contributions to organism survival, which may be because they operate in the same pathway or, alternatively, because they operate in different but functionally intersecting pathways. The other major DNA repair pathway in bacteria is the RecBCD pathway, which primarily targets and repairs double-stranded breaks in DNA (43). Although genes in this pathway did not associate with DUF328 genes in our comparative genomics study, it is possible that YaaA is involved in this or other DNA-maintenance processes that partially overlap with the RecFOR pathway (44). A circumstantial argument against DUF328 proteins being direct participants in the RecFOR pathway is that this DNA repair pathway is present in archaea but DUF328 proteins are rare in that kingdom. Moreover, defects in the RecFOR pathway in E. coli result in phenotypes that do not depend on oxidative stress, whereas a phenotype is seen in ΔyaaA E. coli only in oxidative stress conditions (4). However, the selective protective role of YaaA_Ec during oxidative stress may be driven by its increased expression under oxidative stress, rather than specificity for repair of oxidative DNA damage. Although the details of DUF328 proteins' role in DNA protection during oxidative stress remain to be determined, cofitness analysis indicates a probable contribution from the RecFOR pathway in diverse bacteria.

The crystal structure of YaaA_Ec reveals a new fold for the DUF328 family (45). Given its resemblance to a slice of cantaloupe (Fig. S10), we propose that this be called the cantaloupe fold. This fold features a distinctive abundance of structured peptide stretches that are neither α-helix nor β-strand and an apical cleft demarcated on one end by a HhH DNA-binding domain and on the other by a β-strand motif. HhH domains are nonsequence-specific DNA-binding modules that are commonly found in proteins that digest, synthesize, or repair DNA (39). The apical cleft is highly basic, enriched in conserved residues, and a plausible site for DNA binding. It is unclear if DNA could simultaneously bind both regions and whether such bound DNA would be partially unwound or otherwise structurally perturbed. The minor groove-binding preference of HhH motifs suggests that at least some portion of the bound DNA should be a B-form double helix. The presence of two candidate DNA-binding sites may explain the presence of multiple shifted bands at higher concentrations of protein in the EMSA of YaaA_Ec-DNA complexes (Fig. 3). These multiple bands indicate a YaaA_Ec:DNA binding stoichiometry of greater than 1:1 at higher YaaA_Ec concentrations, consistent with multiple YaaA_Ec proteins binding cooperatively to the DNA. The Hill coefficient of 3.1 for YaaA_Ec binding indicates strong positively cooperative DNA binding, suggesting either that protein-protein interactions enhance YaaA_Ec affinity for DNA or that binding-induced perturbations to DNA structure recruit additional YaaA_Ec molecules. Determining how YaaA_Ec interacts with DNA will be an important future direction for elucidating the molecular basis of DNA recognition by this new protein fold.

DpdC proteins are members of the DUF328 family that possess nitrile hydratase activity involved in PreQ₀ metabolism (9, 10). The involvement of DpdC proteins in PreQ₀-containing DNA is broadly consistent with our results connecting YaaA_Ec to DNA repair, although we find that YaaA_Ec is not involved in queuosine metabolism in E. coli. Regardless, the nitrile hydratase activity of DpdC proteins demonstrates that the DUF328 cantaloupe fold can support enzymatic activity, leaving open the possibility that YaaA_Ec may have an undiscovered enzymatic function. An important avenue for future research will be to determine the mechanism by which YaaA_Ec (and other DUF328) proteins regulate Fe²⁺ levels to protect cells from oxidative stressors (4), including whether YaaA_Ec simply binds DNA or acts on it as a substrate. This report provides a molecular activity for YaaA_Ec and other DUF328 proteins that will inform additional research into the protective mechanisms of this new family and fold class of DNA-binding protein.

Experimental procedures

Bioinformatics analyses

The protein sequences were retrieved from the NCBI Protein Database using the following accession numbers: YaaA_Ec, Uniprot ID P0A8I3; DpdC, AJQ72467.1. Protein domain analysis was performed using HHpred by the MPI Bioinformatics Toolkit (46, 47) against the Pfam database (48) using default parameters.

319 reviewed sequences of the DUF328/IPR005583/PF03883 family were retrieved from InterPro (RRID:SCR_006695). Multiple sequence alignment was built using MAFFT (RRID:SCR_011811) (49). WebLogo (50) was used to generate the sequence logo. Domain architecture was analyzed by the Conserved Domain Architecture Retrieval Tool (51).

SSNs were generated with the Enzyme Function Initiative suite of webtools (16, 17). SSNs were visualized using Cytoscape (52). The parameters used for the generation of the DUF328/IPR005583/PF03883 SSNs were as follows: for the whole family SSN, the input method “FASTA” (option C) was used, using YaaA_Ec and IPR005583, UniRef90 with the minimum length filter of 100 and maximum length filter of 500. The alignment score threshold was 90. Sequences that share 90% or more identity are collapsed together to a single node to reduce the complexity for visualization. The obtained SSN was subjected to Enzyme Function Initiative Genome Neighborhood Tool analysis to obtain genome neighborhood diagrams.

Fitness data were retrieved from the Fitness Browser (RRID:SCR_018981) (23, 53) using YaaA_Ec as the query sequence. Genes that show strong cofitness (cofitness >0.75) and strong conserved cofitness (cofitness >0.6 and ortholog cofitness >0.6) with DUF328 genes are placed in Table 1. Genes with ortholog cofitness >0.6 and cofitness × ortholog cofitness >0.3 with DUF328 genes are placed in Table S1. The AnnoTree v1.2.0 tool (54) was used to analyze the distribution of members of the PF03883 family in the reference species set from GTDB bacteria release RS89 (55).

Bacterial strains

All strains used in this study are listed in Table 2. E. coli strains were routinely grown in LB media (Tryptone 10 g/liter, yeast extract 5 g/liter, and NaCl 5 g/liter) at 37 °C. When antibiotic selection was required, media were supplemented with 100 μg/ml ampicillin, 25 μg/ml chloramphenicol, or 50 μg/ml kanamycin. PreQ₀ was purchased from Ark Pharm.

Table 2.

Strains used in this study

Strain name	Genotype/relevant characteristics	Reference/source
E. coli MG1655	WT	(Liu, Y. et al., 2011)/Imlay laboratory UIUC
ΔyaaA	As MG1655 plus Δ(yaaA1::cat)1	(Liu, Y. et al., 2011)/Imlay laboratory UIUC
ΔqueD	As MG1655 plus ΔqueD::cat	This study
Δtgt	As MG1655 plus Δtgt::cat	H. Mori Collection
ΔqueD ΔyaaA	As ΔyaaA plus ΔqueD::cat	This study
Δtgt ΔyaaA	As ΔyaaA plus Δtgt::cat	This study
Hpx⁻	As MG1655 plus Δ(ahpC-ahpF′) kan::′ahpF Δ(katG17::Tn10)1 Δ(katE12::Tn10)1	(Liu, Y. et al., 2011)/Imlay laboratory UIUC
Hpx⁻ ΔyaaA	As Hpx⁻ plus Δ(yaaA1::cat)1	(Liu, Y. et al., 2011)/Imlay laboratory UIUC
Hpx⁻ ΔqueD	As Hpx⁻ plus ΔqueD::cat	This study
Hpx⁻ ΔyaaA ΔqueD	As Hpx⁻ ΔyaaA plus ΔqueD::cat	This study

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Growth studies were adapted from those previously described (4). Anaerobic overnight cultures were diluted into anaerobic LB and grown for four to five generations to early log phase (optical density at 600 nm (A₆₀₀) of 0.15 to 0.20). The cultures were then diluted into aerobic LB of the same composition to an A₆₀₀ of ≈0.005. All cultures were grown at 37 °C. LB medium was made 1 day prior to culturing and stored in the dark to avoid the photochemical generation of H₂O₂. It was transferred immediately after autoclaving to an anaerobic chamber (BACTRON anaerobic chamber), where it was stored under an atmosphere of 5% CO₂, 10% H₂, and 85% N₂ overnight or longer prior to use.

Expression and purification of YaaA_Ec

The YaaA_Ec gene (yaaA:b0006) was PCR-amplified from E. coli XL1-blue genomic DNA using Platinum II Taq DNA polymerase (Thermo Fisher Scientific) and primers that introduced 5′ NdeI and 3′ XhoI restriction sites. The YaaA_Ec gene was subcloned between the NdeI and XhoI sites of pET15b (Novagen) and sequence-verified by dideoxy DNA sequencing. The validated YaaA_Ec-pET15b construct was transformed by heat shock into chemically competent BL21(DE3) (Novagen) E. coli for protein expression. This construct expresses YaaA_Ec with a thrombin-cleavable N-terminal hexahistidine tag, although the tag was difficult to remove from the purified protein with thrombin and was retained in the final purified protein. BL21(DE3) cells containing YaaA_Ec-pET15b were grown in LB medium with 100 μg/ml ampicillin at 37 °C with shaking at 270 rpm to an A₆₀₀ of 0.2–0.3, at which point the culture was transferred to 20 °C and incubated with shaking at 150 rpm for an additional two h. YaaA_Ec expression was induced with the addition of isopropyl β-d-1-thiogalactopyranoside (Calbiochem) to a final concentration of 0.2 mm, and the culture was incubated at 20 °C with shaking overnight. Chloramphenicol was added to a final concentration of 100 μg/ml two h prior to harvest to enhance protein solubility (56). Cells were harvested by centrifugation and cell pellets were frozen on liquid N₂ and stored at −80 °C.

Recombinant hexahistidine-tagged YaaA_Ec was purified using nickel-nitrilotriacetic acid metal affinity chromatography as previously described (57). Briefly, the cell pellet was lysed by the addition of lysozyme to a final concentration of 1 mg/ml followed by sonication. The crude lysate was clarified by centrifugation at 12,000 × g and the supernatant was incubated with HIS-select nickel-nitrilotriacetic acid resin (Sigma-Aldrich), washed with wash buffer (25 mm HEPES, pH 7.5, 300 mm NaCl, and 25 mm imidazole), and eluted with elution buffer (25 mm HEPES, pH 7.5, 300 mm NaCl, and 250 mm imidazole). Fractions containing the YaaA_Ec protein were determined by Coomassie-stained SDS-PAGE and the purest fractions were pooled. Despite bearing a thrombin cleavage site downstream of the N-terminal hexahistidine tag, incubation of the purified protein with thrombin did not efficiently cleave the hexahistidine tag. Therefore, the final protein retains the tag and the thrombin cleavage site, adding the N-terminal sequence MGSSHHHHHHSSGLVPRGSH before the first methionine of YaaA_Ec.

YaaA_Ec co-purified with large amounts of nucleic acid that was identified as DNA based on its sensitivity to DNaseI on GelRed (Biotium)-stained agarose gel electrophoresis. The contaminant DNA was present at ∼0.1 mg/mg of protein based on 260 nm/280 nm absorbance ratio and could not be effectively removed by passage over a High Q anion exchange column (Bio-Rad). The large majority of the DNA could be removed using hydroxyapatite chromatography (ceramic hydroxyapatite Type II resin, Bio-Rad) with gradient elution from 20 to 500 mm KPO₄ buffer, pH 7.2, over 10 column volumes. A small amount of residual nucleic acid remained in YaaA_Ec even after hydroxyapatite chromatography as determined by GelRed-stained agarose gel electrophoresis of the purified protein. After hydroxyapatite chromatography, the final YaaA_Ec protein was dialyzed into storage buffer (25 mm Tris-HCl, pH 8.8, and 100 mm KCl) and was concentrated to 28 mg/ml (ε₂₈₀ = 25,900 m⁻¹ cm⁻¹) with a 10-kDa molecular weight cutoff regenerated cellulose membrane spin concentrator at 4 °C (Millipore). Before storage, 10 mm EDTA (final concentration) was added to enhance protein stability and the protein was flash-frozen in 50–200-μl aliquots in liquid N₂ and stored at −80 °C until needed.

YaaA_Ec DNA-binding assay

Synthetic Holliday junction (X-0), single-stranded, and double-stranded oligonucleotide substrates were generated as previously described (58, 59). DNA-binding assays were performed by incubating YaaA_Ec protein with 4 pg of ³²P-labeled oligonucleotide substrates in DNA-binding buffer (30 mm HEPES, pH 7.5, 1 mm DTT, and 100 μg/ml BSA) on ice for 15 min. The protein-DNA complexes were analyzed on 5% native polyacrylamide gels. To compare YaaA_Ec binding to either circular DNA, blunt-end linear DNA, or linear DNA with single-stranded overhangs, 0.5 μg of pUC19 plasmid (Thermo Fisher Scientific, SD00661) with or without SmaI or SalI digestion was incubated with the indicated amount of YaaA_Ec in DNA-binding buffer on ice for 30 min prior to separation of protein-DNA complexes on 0.6% agarose gels. ImageJ (60) was used to quantify the band intensities to determine the relative amount of DNA that was free or in complex with YaaA_Ec. These fractional binding values were plotted as a function of total YaaA_Ec concentration and fit to a single-site binding model with positive cooperativity in Prism (GraphPad) to determine the K_D and the Hill coefficient. DNA binding was assayed for multiple independent preparations of recombinant YaaA_Ec to ensure consistency.

X-ray crystallographic structure determination of YaaA_Ec

Crystallization conditions for purified hexahistidine-tagged YaaA_Ec at 28 mg/ml in storage buffer were screened using commercial sparse matrix screens in sitting-drop 96-well plates. Protein and reservoir solutions were dispensed in 0.5-µl drops using a Gryphon liquid handling robot (Art Robbins Instruments). Initial needle-shaped crystals were optimized by manual screening using sitting-drop vapor equilibration and improved crystals grew in 100 mm NaCl, 100 mm sodium citrate, pH 4.6, 100 mm Na₂HPO₄, 75 mm NaH₂PO₄, and 15% PEG 8000. Further optimization with the Hampton additive screen identified 3% benzamidine-HCl as improving crystal morphology and size. An ordered benzamidine molecule makes hydrogen bonds to two Pro⁷³ residues related by crystallographic symmetry in the final structure. In addition, the phenyl ring of benzamidine participates in cation-π interactions with two symmetry-related Arg⁷⁷ residues, explaining why this additive aided the growth of diffraction-quality crystals.

Because YaaA_Ec has no homologs of known structure, experimental phasing using SeMet SAD was used to calculate initial electron density maps. The pET15b-YaaA_Ec expression construct was transformed into the methionine auxotroph E. coli strain B834(DE3) (Novagen) by heat shock of chemically competent cells. YaaA_Ec was expressed in M9 minimal medium supplemented with 42 mg/liter of each l-amino acid except methionine and cysteine, 125 mg/liter each of adenine, guanosine, thymine, and uracil, 4 mg/liter thiamin, 4 mg/liter d-biotin, and 30 mg/liter l-selenomethionine (Acros Organics). SeMet-YaaA_Ec was purified as described above and crystallized in the same condition as the native protein. Diffraction-quality crystals of SeMet-YaaA_Ec were cryoprotected by serial transfer and brief soaking in reservoir solution supplemented with ethylene glycol in 5% increments to a final concentration of 15%. Crystals were mounted in nylon loops and cryocooled by rapid immersion in liquid nitrogen.

YaaA_Ec crystallized in space group P2₁2₁2₁ with two protein chains in the asymmetric unit. Diffraction data extending to a 1.65-Å resolution were collected from a SeMet-YaaA_Ec crystal measuring 350×100×100 μm at beam line 7-1 of the Stanford Synchrotron Radiation Lightsource using the oscillation method. The incident X-rays were tuned to the K-edge of selenium (0.9788 Å) to maximize anomalous signal from the six SeMet residues in YaaA_Ec. Inverse beam geometry was not used. Data were processed using HKL2000 (61), and significant anomalous signal was extended to a 2.45-Å resolution using a CC_anom cutoff of 0.15 (62) as determined by Aimless (63) in the CCP4 suite (64). See Table S2 for data statistics.

SAD phasing using unmerged input reflections and local scaling was performed in PHENIX (65). The figure of merit for the initial experimental SAD phases was 0.39, and these phases were improved by density modification prior to autobuilding in PHENIX (66, 67). The initial autobuilt model was manually improved in Coot (68), including addition of ordered waters. Five residues of the uncleaved N-terminal tag were ordered in the crystal and thus included in the model. Refinement was performed in PHENIX using a maximum likelihood target function based on anomalous amplitudes (i.e. Bijvoet mates were kept separate), optimization of the X-ray/stereochemical and ADP weights, and translation-libration-screw treatment of the ADPs (66). Benzamidine was modeled into unambiguous electron density and mediates a key crystal-packing contact, explaining why it was an effective crystallization additive. Final model validations were performed using Coot (68) and MolProbity (69). The final model has excellent stereochemical and clashscore statistics, with an overall MolProbity score of 0.95 (100^th percentile). See Table S2 for model statistics.

YaaA_Ec structural analysis and display

The surface electrostatic potential of YaaA_Ec was calculated using the APBS (30, 70) and using PDB2PQR (71) for atomic partial charge and radius assignments. Default values of solvent (78) and protein (2) dielectric constant, probe radius (1.4 Å), and temperature (298.15 K) were used. Structural figures were made with UCSF Chimera (72) and POVScript+ (73). Sequence conservation was mapped onto the structure of YaaA_Ec using the ConSurf server (74).

Data availability

Data for Fig. 1, Fig. 2, and Fig. S1 are available from Dr. Valérie de Crécy-Lagard, University of Florida, vcrecy@ufl.edu. Data for Fig. 3, Table 1, Figs. S3–S7, Fig. S12, and Table S1 are contained in their entirety in the article. Data for PF03883 sequence similarity network and data for growth curves are available at University of Florida Digital Collections: Fig. S2 (https://ufdcimages.uflib.ufl.edu/IR/00/01/12/21/00001/PF03883_SSN.xlsx), Fig. S8 (https://ufdcimages.uflib.ufl.edu/IR/00/01/12/21/00001/Data%20for%20FigureS8.xlsx), and Fig. S9 (https://ufdcimages.uflib.ufl.edu/IR/00/01/12/21/00001/Data%20for%20FigureS9.xlsx). X-ray crystallographic structure factor data and coordinates (Figs. 4 –6, Fig. S10, Fig. S11, and Fig. S13) are available for download from the PDB (5CAJ). Raw diffraction data are available by request from Dr. Mark Wilson, University of Nebraska, mwilson13@unl.edu.

Supplementary Material

Supporting Information

supp_295_41_14236__index.html^{(994B, html)}

Acknowledgments

We thank Professor James Imlay and Dr. Yuanyuan Liu (University of Illinois) for the gift of the YaaA deletion and the Hpx⁻ catalase- and peroxidase-deficient E. coli strains and Carol Cook for photography.

This article contains supporting information.

Author contributions—J. P., Y. Y., J. L., V. dC.-L., and M. A. W. data curation; J. P., Y. Y., Y. L., V. dC.-L., and M. A. W. formal analysis; J. P., Y. Y., C.-W. C., V. dC.-L., and M. A. W. validation; J. P., Y. Y., J. L., C.-W. C., D. I.-R., Y. L., V. dC.-L., and M. A. W. investigation; J. P., Y. Y., Y. L., V. dC.-L., and M. A. W. methodology; J. P., Y. Y., C.-W. C., Y. L., V. dC.-L., and M. A. W. writing-original draft; J. P., Y. Y., J. L., C.-W. C., D. I.-R., Y. L., V. dC.-L., and M. A. W. writing-review and editing; Y. Y., V. dC.-L., and M. A. W. visualization; Y. L., V. dC.-L., and M. A. W. supervision; Y. L., V. dC.-L., and M. A. W. funding acquisition; V. dC.-L. and M. A. W. conceptualization; V. dC.-L. and M. A. W. resources; V. dC.-L. and M. A. W. project administration.

Funding and additional information—Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by the National Institutes of Health, NIGMS Grant P41GM103393. Portions of this research were also funded by the National Institutes of Health Grants R01GM70641 (to V. dC.-L.). and R01GM092999 (to M. A. W.). Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH Grant P41GM103311. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or the National Institutes of Health.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

dADG: 2′-deoxy-7-amido-7-deazaguanosine
dPreQ₀: 2′-deoxy-7-cyano-7-deazaguanosine
HhH: helix-hairpin-helix
GTDB: Genome Taxonomy Database
SSN: sequence similarity network
GGN: genome neighborhood network
TGT: tRNA guanine transglycosylase
SAD: single-wavelength anomalous diffraction
SeMet: selenomethionine
APBS: Adaptive Poisson-Boltzmann Solver
ADP: atomic displacement parameter.

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Supplementary Materials

Supporting Information

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Data Availability Statement

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PERMALINK

The DUF328 family member YaaA is a DNA-binding protein with a novel fold

Janani Prahlad

Yifeng Yuan

Jiusheng Lin

Chou-Wei Chang

Dirk Iwata-Reuyl

Yilun Liu

Valérie de Crécy-Lagard

Mark A Wilson

Abstract

Results

Comparative genomic analyses show that DUF328 genes are widespread and are fused or physically clustered with genes involved in oxidative stress and DNA repair

Figure 1.

Figure 2.

DUF328 genes show strong cofitness with the RecFOR pathway for DNA repair

Table 1.

Recombinant YaaAEc binds DNA with nanomolar affinity

Figure 3.

YaaAEc role is PreQ0-independent

YaaAEc possesses a new fold

Figure 4.

Figure 5.

YaaA contains a HhH DNA-binding motif

Figure 6.

Discussion

Experimental procedures

Bioinformatics analyses

Bacterial strains

Table 2.

Expression and purification of YaaAEc

YaaAEc DNA-binding assay

X-ray crystallographic structure determination of YaaAEc

YaaAEc structural analysis and display

Data availability

Supplementary Material

Acknowledgments

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Recombinant YaaA_Ec binds DNA with nanomolar affinity

YaaA_Ec role is PreQ₀-independent

YaaA_Ec possesses a new fold

Expression and purification of YaaA_Ec

YaaA_Ec DNA-binding assay

X-ray crystallographic structure determination of YaaA_Ec

YaaA_Ec structural analysis and display