Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3′ End of Structured DNA in Repair Pathways

Structure analysis of the exonuclease RNase T reveals that it also functions in DNA repair pathways where it binds and processes bulge, bubble, and Y-structured DNA to trim the DNA 3′ ends.


Introduction
It is well known that DNA repair mechanisms maintain genomic integrity and are essential for cell survival. Damaged DNA can be restored by a variety of DNA repair processes, such as direct reversal, base excision, nucleotide excision, mismatch, and recombination repair pathways [1]. Although diverse proteins play different roles in these pathways, DNA repair is generally accomplished by a coordinated effort via several types of DNA enzymes, including endonucleases that nick DNA near the damaged site, exonucleases that trim DNA from the broken end, helicases that unwind duplex DNA, polymerases that make new strand DNA with correct sequences, and ligases that seal the restored DNA strands. Among all these DNA enzymes, the molecular functions of exonucleases, which bind at the 39 or 59 end of DNA and cleave one nucleotide at a time, are least understood. How they select, rather than randomly bind to, a broken end of DNA and process it up to the site for the next-step processing remains to be investigated.
Here we use the bacterial exonuclease RNase T as a model system to study the processing of DNA in various DNA repair pathways. RNase T is a member of the DnaQ-like 39-59 exonucleases with a DEDDh domain that contains four acidic DEDD residues (D23, E25, D125, and D186) for binding of two magnesium ions, and one histidine residue (H181) for functioning as the general base in the active site for the hydrolysis of the 39-terminal phosphodiester bond of a nucleic acid chain [2]. The family of DnaQ-like exonucleases constitutes thousands of members, all with exonuclease activity either processing RNA during RNA maturation, interference, and turnover, or processing DNA during DNA replication, degradation, repair, and recombination. A number of the DnaQ-like exonucleases have been shown to play a role in DNA repair. Usually the DEDDh domain can be linked to a DNA polymerase domain for proofreading during DNA replication, such as the DnaQ domain of the e subunit of E. coli pol III holoenzyme and the exonuclease domain of human pol d, e, and c [3]. Mutations in or deletion of the proofreading 39 exonuclease domain for these polymerases are either lethal or induce high mutation rates and high incidence of cancers [4]. The DEDDh domain can also be linked to a helicase domain and functions in processing of broken DNA strands during DNA repair and recombination, such as that of human WRN [5]. Mutations in the DEDDh exonuclease domain of WRN are associated with Werner syndrome that results in premature aging and increased risk of cancer [6].
However, most of the DEDDh domain functions as an autonomous protein and is not linked to a polymerase or a helicase domain. Some of these exonucleases participate in DNA 39-end processing in DNA repair, such as ExoI and ExoX from E. coli and TREX1 and TREX2 from human [7][8][9]. ExoI and ExoX are monomeric enzymes that digest single-stranded DNA in mismatch and DNA recombination repair pathways [10][11][12], whereas the human TREX1 and TREX2 are dimeric enzymes, likely processing single-stranded DNA in mammalian cells [13,14]. Mutations in TREX1 are linked to the autoimmune diseases Aicardi-Goutieres syndrome and systemic lupus erythematosus, probably due to the accumulation of nonprocessed intermediate DNA during replication and repair pathways [15][16][17]. The crystal structures of ExoI [18], TREX1 [19], and TREX2 [20] reveal that they all bear a classical a/b fold of the DEDDh domain; nevertheless, their precise functions in DNA processing remain uncertain.
RNase T has also been implicated in the UV-repair pathways based on the observations that the cells lacking RNase T are less resistant to UV radiation and overexpression of RNase T can rescue the UV sensitivity of the ExoI knockout E. coli strain [21,22]. Yet RNase T was originally recognized as an RNase based on its indispensable role in tRNA 39-end processing during tRNA maturation [23]. RNase T also performs the final trimming for various stable RNA, including 5S and 23S rRNA [24,25]. RNase T can digest both DNA and RNA and it has a unique specificity that its exonuclease activity is reduced by a single 39terminal C or completely abolished by a dinucleotide 39-terminal CC in digesting either DNA or RNA, referred to as the C effect [26]. Moreover, its exonuclease activity is inhibited by duplex structures, referred to as the double-strand effect; therefore, a 39 overhang of a duplex DNA or RNA is only digested near the duplex region by RNase T [26,27]. Previous crystal structures of RNase T in complex with various single-stranded DNA (39terminal G versus C) and double-stranded DNA (1 versus 2 nucleotide 39 overhang) reveal the structural basis for C effect and double-strand effect [27,28]. The binding of an uncleavable substrate, such as a single-stranded DNA with a 39-terminal C or a duplex DNA with a short 2-nucleotide 39 overhang, induces an inactive conformational change in the active site and thus inactivates the exonuclease activity. Therefore, in digesting a duplex DNA with a 39 overhang, RNase T can accurately differentiate its cleavable or noncleavable substrates based on the C effect and double-strand effect, and it produces a precise final product of a duplex with a 1-nucleotide (if the last base pair in duplex region is AT) or 2-nucleotide (if the last base pair in duplex region is GC) 39 overhang if the CC dinucleotide is not present within the 39 tail, or else it stops at the 39-CC end. RNase T hence is capable to trim various precursor RNA to produce mature RNA with a precise 39 overhang depending on the structure and sequence of these precursors: 1 nt for 5S rRNA, 2 nt for 23S rRNA, 4 nt for 4.5S RNA, and 4 nt for tRNA [27,28].
In fact, RNase T is a more efficient DNase than RNase in that it digests DNA with a 10-fold efficiency as compared to RNA (see figure S2 in [28]), supporting its possible cellular role in DNA processing. However, it is not certain if RNase T indeed processes DNA in DNA repair, and if it does, how it selects and processes its DNA substrates. To determine the molecular function of RNase T, we show here by biochemical and structural approaches that it is a structure-specific DNase capable of digesting intermediate structured DNA during DNA repair. We found that RNase T not only digests bubble and bulge DNA in Endonuclease V (Endo V)dependent DNA repair but also digests Y-structured DNA in UVinduced DNA repair pathways. The crystal structures of RNase T in complex with a bulge and a Y-structured DNA further demonstrate how this dimeric enzyme elegantly binds and processes these structured DNA molecules in different ways. Our results reveal, for the first time, the precise molecular role of an exonuclease in the 39 end DNA processing and may hint at the molecular function for other members of DnaQ-like exonucleases.

RNase T-Deficient Cells Are Sensitive to DNA Damaging Agents
To confirm the possible roles of RNase T in DNA repair, we measured the chronic and acute sensitivity of the RNase T knockout E. coli strain (Drnt)?against various DNA-damaging agents, including hydrogen peroxide (H 2 O 2 ), methyl methanesulfonate (MMS), 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), and UV light. A number of exonucleases that have been shown to play a role in DNA repair, including ExoI (DexoI) [7,10], ExoX (DexoX) [7], PNPase (Dpnp) [29], and RecJ (Drecj) [30], were tested in parallel for a comparison (Table 1 and Figures S1 and S2). The wild-type K-12 strain resisted all DNA-damaging agents when present at a chronic dose, whereas RNase T-deficient strain (Drnt) had a slow growth phenotype and was sensitive to the chronic dose of H 2 O 2 , MMS, 4NQO, and UV-C ( Figure 1A). The RNase T-deficient strain (Drnt) was also sensitive to the acute dose of H 2 O 2 in various concentrations from 20 to 80 mM ( Figure 1B). The sensitivity of Drnt strain to UV-C was different from those observed in the previous report [21]; therefore, we further confirmed the UV and H 2 O 2 sensitivity by rnt-rescued experiments, which restored the resistance of Drnt cells against UV-C and H 2 O 2 (see Figure S1). This result shows that the sensitivity of the RNase T knockout cells to UV-C and H 2 O 2 is indeed due to the deficiency of RNase T. H 2 O 2 produces a wide variety of DNA lesions, including singlestrand/double-strand breaks (DSB), oxidation and deamination of bases, and sugar modifications [31,32], that are usually restored by direct repair (DR), base excision repair (BER), and alternative repair (AR) [1,29,30,33]. DNA alkylating agent MMS produces Author Summary DNA repair relies on various enzymes, including exonucleases that bind and trim DNA at broken ends. However, we know little about how an exonuclease precisely selects and trims a DNA broken end in specific repair pathways. In this study, the enzyme RNase T, previously known for its involvement in processing RNA substrates, is shown to also possess DNase activity. RNase T is a DnaQ-like exonuclease and is characterized in this work as the exoDNase responsible for trimming the 39 ends of structured DNA in various DNA repair pathways. Based on the high-resolution crystal structures of RNase T-DNA complexes, an insightful working model is provided showing how RNase T processes bulge, bubble, and Ystructured DNA in various DNA repair pathways. RNase T thus represents a unique structure-specific exonuclease with multiple functions not only in processing 39 overhangs of duplex RNA during RNA maturation, but also in processing 39 ends of bubble, bulge, and Ystructured DNA during DNA repair. These findings advance our understanding of the precise function of an exonuclease in DNA repair and suggest possible roles for thousands of members of DnaQ superfamily exonucleases in DNA repair and replication.
methylated DNA bases that can be restored by DR and BER [34,35]. MMS also leads to the accumulation of single-strand gaps (SSGs) and DSB-related DNA damage [29,35]. MMC is a DNA cross-linking agent that can trigger the SOS response and creates damage repaired by NER [29,36,37]. UV light-mimetic agent 4NQO can produce replication-blocked DNA base adducts, SSGs, and DSB-related DNA damages [29,38] that are mainly repaired by NER [38]. UV-C irradiation (100-290 nm) leads to three major base modifications and DSB-related DNA damage that are usually repaired by BER and DNA recombination [31,39,40]. The sensitivity of the Drnt strain to H 2 O 2 , MMS, 4NQO, and UV-C suggests that RNase T may play a role in BER, AR, and DSBrelated DNA repair pathways.
In comparison to the known DNA-repair exonucleases, RNase T had a wider sensitivity to various DNA-damaging agents. The ExoI-deficient cells were only sensitive to H 2 O 2 ; the ExoXdeficient cells were not sensitive to any DNA-damaging agents; the PNPase-deficient cells were sensitive to H 2 O 2 , MMS, and UV-C; and the RecJ-deficient cells were sensitive to 4NQO and UV-C (Table 1 and Figure S1). RNase T had a more apparent and wider sensitivity as compared to those of ExoI, ExoX, PNPase, and RecJ, suggesting that RNase T plays more extensive and crucial roles in various DNA repair pathways.
To further characterize the role of RNase T in DNA repair pathways, the single-stranded DNA containing a methylated, deaminated, or oxidized base, or an abasic site at the 39-terminal end  were incubated with RNase T in the DNA digestion experiment. We found that RNase T digested the DNA with a methylated base-O 4 -methylthymine (O 4 -mT) and O 6 -methylguanine (O 6 -mG)-and a deaminated base-uracil and hypoxanthine. However, the DNA with a 39-terminal oxidized base, 8-oxoguanine (8-oxo), and an abasic site were more resistant to RNase T digestion (see Figure 1C). This result suggests that RNase T can function as an exonuclease in the excision step for methylated and deaminated bases in BER and AR.

RNase T Processes Bulge and Bubble DNA Without Sequence Preference
The next question we tackled was what types of DNA that can be processed by RNase T in DNA repair, besides the singlestranded DNA with a lesion. RNase T is not an appropriate exonuclease for digesting single-stranded DNA since its exonuclease activity is easily blocked by any C within a DNA strand. A variety of intermediate structured DNAs are generated during DNA repair, such as bulge, bubble, and Y-structured DNA. Bulge DNAs are produced in frameshift DNA mutations during DNA replication of repetitive sequences [41], whereas bubble DNA are generated in mismatch replication or deamination of DNA bases [42]. Y-structured DNAs are generated in various DNA repair pathways, such as mismatched DNA repair and DNA recombination (see Discussion). To test if RNase T processes these intermediate structured DNA, we incubated RNase T with different DNA and found that RNase T can digest Y-structured DNA and blunt-end bubble DNA with an I-T or I-G bubble ( Figure 2A). In digesting the Y-structure DNA, the exonuclease activity of RNase T was blocked by the duplex structure-that is, double-strand effect-and RNase T generated a final product of 1nucleotide 39 overhang duplex ( Figure 2A). In digesting bulge and bubble DNA, the double-strand effect did not occur, and the blunt-end bulge and bubble DNA was cleaved by RNase T (Figure 2A).
We further tested the sequence preference of RNase T in digesting the structured DNA. In digesting a classical duplex DNA with a short 39 overhang, the exonuclease activity of RNase T was blocked by a dinucleotide 39-end CC sequence ( Figure 2B). In contrast to the duplex DNA, the bulge and Y-structured DNA with terminal 39-CC were processed by RNase T into a final product with a 1-nucleotide 39 overhang ( Figure 2B). Therefore, in digesting bubble and bulge DNA, RNase T has no sequence preference, and it removes the last paired nucleotide of any sequence to generate a 1-nucleotide 39 overhang. In digesting Ystructured DNA, RNase T also has no sequence preference and processes the 39 tail of any sequence close to the duplex region to generate a 1-nucleotide 39 tail as the final product.

Crystal Structure Reveals How RNase T Binds and Processes Blunt-End Bulge DNA
We were intrigued by how RNase T could bind and process a bubble or bulge in DNA with a blunt end. Previous studies showed that the double-strand effect of RNase T requires a 39 overhang of a duplex with a length of more than 2 nucleotides for inserting into the active cleft for digestion (see Movie S1). To reveal how RNase T binds and processes a DNA bulge with a blunt end, we cocrystallized RNase T with two bulge DNA molecules, one with a 39-end TC and one with a 39-end CC sequence in acidic conditions, pH 5.5 and pH 6.0, respectively (see Table S2). RNase T only digests nucleic acids in basic conditions because the general base H181 has to be deprotonated to activate a nucleophilic water for hydrolysis. Therefore, due to the low pH, the bulge DNA in the crystal were not cleaved by RNase T. The crystal structure of the two complexes was solved by X-ray diffraction methods at a resolution of 1.8 and 2.0 Å , respectively (see Figure 3). In the RNase T-bulge DNA complex structures, the dimeric RNase T bound to two bulge DNAs, with the 39 end of DNA binding at the active site of each protomer ( Figure 4). The bulge DNA was bound between the two RNase T protomers, in a way similar to that of the classical duplex DNA with a 39 overhang [28]. However, in contrast to the previous duplex DNA complex, the aromatic side chain of Phe29 was inserted into the bulge and stacked with the two neighboring GC base pairs in both of the bulge DNA complexes (see Figure 4A and 4B). In the previous duplex DNA complex, Phe29 was stacked with the 59-end base of the opposite nonscissile strand, and the stacking stopped the further cleavage of the scissile strand at the 39 end, resulting in the double-strand effect (see the schematic comparison in Figure 4C). The crystal structure of the bulge DNA complex revealed how RNase T can overcome the double-strand effect by inserting Phe29 into the bulge so that the 39-end scissile phosphate was moved accordingly into the active site (see Movie S2). We found that the active site of the bulge DNA complex indeed had an active conformation with two bound Mg 2+ ions, and the general base His181 was located close to the scissile phosphate ( Figure 4B).
The crystal structure of the bulge DNA complex also revealed how RNase T could overcome the C effect. The 39-end cytosine was paired with the 59-end guanine, and this base pairing prevented Glu73 from interacting with the 39-end C to induce the C effect ( Figure 2B, Figure S2B). Therefore, the bulge DNA could be processed by RNase T without any sequence preference. Moreover, the 59 end of bulge DNA was not hindered by any residue and could further extend ( Figure S2A), suggesting that RNase T can cleave bulge DNA with a long single-stranded region at the opposite strand, similar to those DNA in the frameshift DNA mutations (see Discussion) [41]. The crystal structure thus reveals at the atomic level how RNase T binds and processes a bulge DNA with a blunt end without a sequence preference.

RNase T Is Likely a Downstream Exonuclease That Follows Endo V Nicking
The bubble and bulge DNA can be produced by Endo V, which makes a nick at the 39 side one base pair away from a damage site with a deaminated base in the alternative DNA repair [42]. Endo V also processes mismatched DNA, hairpin-containing DNA, bulge DNA, and flap DNA [43][44][45], however the downstream process following Endo V nicking has not been characterized. The bulge DNA in our crystal structures had a conformation similar to the bubble DNA produced by Endo V nicking, suggesting that RNase T might be the downstream exonuclease of Endo V, responsible for removing the last base-paired nucleotide at the 39 end to release the single-stranded DNA or the damaged DNA bases, such as hypoxanthine, xanthine, and uracil ( Figure 2D).
To test this possibility, we prepared the hypoxanthine-containing-that is, inosine-containing-heteroduplex DNA for examination of Endo V-dependent inosine excision repair in vitro [46].
The heteroduplex DNA plasmid contained the I-G base pair with an AlwNl cutting site and a potential XhoI cutting site. Once the inosine was restored to cytosine, the plasmid could be cleaved by AlwNl and XhoI into two linear double-stranded DNA molecules of 4.1 and 3.1 kilobases ( Figure 2D). The I-G-containing plasmid was then incubated with Endo V, RNase T, ligase, and the Klenow fragment exo 2 (Polymerase I Klenow fragment with a defected 39-59 exonuclease activity). The inosine in the plasmid was restored to cytosine with a higher repair efficiency (86.7%) as compared with those incubated with the wild-type DNA Polymerase I with a proofreading exonuclease domain (61.4%) ( Figure 2C). The repair efficiency was positively correlated with the RNase T concentration and the time of incubation ( Figure S3). Interestingly, ExoI and ExoX could not work with Endo V to restore the inosine to cytosine (unpublished data). These results show that RNase T can work with Endo V in the Endo Vdependent DNA repair.

Crystal Structure Reveals How RNase T Processes Y-Structured DNA in a Unique Way
Beside bubble/bulge DNA, RNase T also processed Ystructured DNA, which can be generated during various DNA repair pathways, such as mismatch repair and DNA recombination. However, it remained unknown how an exonuclease can specifically process the 39-end tail of the intermediate Y-structured DNA. To reveal how RNase T binds and processes a Y-structured DNA, we co-crystallized RNase T with a Y-structured DNA and solved the complex crystal structure at a resolution of 1.9 Å  Table S1). (B) RNase T could not digest a duplex DNA with a 39-CC overhang. However, RNase T digested a bulge DNA and a Y-structured DNA with a 39-CC sequence. The 39 tails are numbered and the corresponding cleavages are indicated by arrowheads on the right of the gel. (C) Endo V-dependent DNA repair is initiated by Endo V cleavage at the 39 side of the second phosphodiester bond to the DNA lesions, such as deoxyriboinosine and insertion/deletion DNA. The mispaired I-T site has a bubble DNA structure, whereas the insertion/deletion DNA has a bulge DNA structure. (D) RNase T worked with Endo V to restore an inosine to cytosine. The heteroduplex DNA plasmid contained a mispaired I-G that could be cleaved by XhoI and AlwNI into two linear 4.1-and 3.1-kb fragments if I-G was repaired to C-G. This heteroduplex plasmid was repaired more efficiently (86.7%) following incubation with Endo V, RNase T, KF exo 2 (the DNA polymerase I Klenow fragment with defective exonuclease activity), and ligase than incubation with Endo V, DNA polymerase I, and ligase (61.4%). doi:10.1371/journal.pbio.1001803.g002 ( Figure 3 and Figure 5). In the crystal structure, the Y-structured DNA was bound to RNase T in a unique way, different from those of the bulge DNA and the duplex DNA that were bound between the two protomers with one strand of DNA bound to one protomer (see Figure 4C). In contrast, both strands of the Ystructured DNA were bound to a single protomer, one Y- structured DNA bound to protomer A and the other DNA molecule bound to protomer B ( Figure 5). This unique binding mode can avoid the hindrance produced by Phe29, which might stack with the 59-end base of the opposite nonscissile strand if the Y-structured DNA was bound in a way similar to that of a duplex DNA. Therefore, in this complex, the opposite nonscissile strand of the Y-structured DNA rotated about 180u to interact with the same protomer of RNase T (see Figure 5B). Several residues, including Gln169, Asp174, Phe175, and Ser177, interacted with the nonscissile strand forming hydrogen bonds with the first and second phosphates in the 59-overhang region, making it fit snugly onto the molecular surface of RNase T ( Figure S5).
The 39 tail of the Y-structured DNA in the crystal structure had a dinucleotide 39-CC sequence. However, the 39-CC did not induce the C effect and inhibit the exonuclease activity of RNase T. A close look at the crystal structure of the Y-structured DNA complex showed that the 39-end C did not interact with Glu73 as it did in the duplex complexes (left panel in Figure S4B).
Moreover, the scissile phosphate of the 39-end C did not shift away from the active site, and as a result, two Mg 2+ ions were bound in the active site in an active conformation (right panel in Figure S4B). Therefore, due to the unique binding mode, the C effect did not occur when RNase T was bound to a Y-structured DNA with a 39-end CC. In summary, this crystal structure reveals how RNase T binds a Y-structured DNA in a unique way and how it processes the 39 tail of any sequence close to the duplex region (see Movie S3).

RNase T Rather Than ExoI and ExoX Trims Structured DNA Near the Duplex Region
Besides RNase T, two monomeric DnaQ-like exonucleases ExoI and ExoX also process DNA during DNA repair in E. coli. ExoI is suggested to play a role in BER [47], mismatch repair [7,10,48,49], UV-related repair [41,50], and DNA replication [51]. ExoX is involved in mismatch repair [11,12] and UV-related inserts into the bulge to stack with the neighboring guanine bases. The magnified view in the right bottom panel shows that the active site of RNase T in the complex has an active conformation with two bound magnesium ions. The light blue balls are water molecules. (C) Schematic diagram of three different binding modes for RNase T bound to a bulge DNA (this study, PDB ID codes 4KB0 and 4KB1), a duplex DNA with a short 39 overhang (previous study [26,27], PDB ID codes 3NH2 and 3VA3), and a Y-structured DNA (this study, PDB ID code 4KAZ). For clarity, only one of the two DNA molecules bound to RNase T is shown. See Movies S1 and S2. doi:10.1371/journal.pbio.1001803.g004 repair [8]. ExoI binds and cleaves long single-stranded DNA [52], whereas ExoX digests both single-stranded and double-stranded DNA [8]. The exonuclease activity of RNase T, ExoI, and ExoX probably overlap and are redundant in these pathways or they may target different substrates. To compare the substrate preference of RNase T to those of ExoI and ExoX, we further expressed and purified ExoI and ExoX for DNA digestion assays. The dynamic light scattering confirmed that ExoI and ExoX were monomeric proteins in contrast to RNase T, which existed as dimeric proteins ( Figure S5).
We found that RNase T, ExoI, and ExoX digested singlestranded 11-nucleotide DNA with similar efficiencies ( Figure 6A). However, in digesting Y-structured DNA with a short 39 overhang, only RNase T and ExoX could process the 39 overhang close to the duplex region, whereas ExoI did not digest Ystructured DNA at low concentrations but did digest Y-structured DNA randomly into small nucleotides at high concentrations ( Figure 6B). In digesting duplex DNA with a short 39 overhang, RNase T processed DNA into a specific length close to the duplex region, generating a final duplex product with a 1-nt 39 overhang at low concentrations ( Figure 6C). ExoX also digested duplex substrates but was less specific, generating various end products with 39 overhangs of different lengths. On the contrary, ExoI could not digest the duplex substrates at the low concentration (0.02 mM) ( Figure 6C). At the high exonuclease concentrations (0.1 and 1 mM), both ExoI and ExoX digested the duplex DNA substrates in the single-stranded and double-stranded regions into small nucleotides. However, RNase T still retained its specificity, only cleaving in the 39 overhang but not in the duplex region ( Figure 6C). These results suggest that RNase T is a highly specific exonuclease that targets the 39 overhang of structured DNA and produces a precise final product. On the other hand, ExoX is less specific and generates 39 overhangs of different lengths in digesting duplex substrates with 39 overhangs, whereas ExoI is specific for single-stranded DNA.
Besides DNA digestion assays, the gel shift assays further showed that RNase T bound with similar affinities to singlestranded DNA, duplex DNA with 4-, 6-, and 10-nucleotide 39 overhangs ( Figure S6). In contrast, ExoI had lower binding affinity for duplex DNA with short 39 overhangs, such as 4 and 6 nucleotides, in agreement with its low activity for these substrates. ExoX also preferred to bind to single-stranded DNA, but not duplex DNA with short 39 overhangs at similar concentrations ( Figure S6). Combining these results of the exonuclease activity and DNA-binding assays, we conclude that RNase T is the ideal exonuclease for trimming the 39 overhang of structured DNA closely to the duplex region, including Y-structured DNA and duplex DNA, whereas ExoI and ExoX mainly process singlestranded DNA in DNA repair.

RNase T Digests Structured DNA in Endo V-Dependent DNA Repair
Our results suggest that RNase T is likely involved in the Endo V-dependent DNA repair pathway. Endo V is a conserved endonuclease playing critical roles in maintaining genome stability in prokaryotes and eukaryotes [53]. Endo V recognizes bubble DNA with mismatched base pairs and deaminated DNA lesions and initiates the Endo V-dependent DNA repair pathway that is independent of BER and MMR [42,44,45,53,54]. Moreover, Endo V nicks frameshift and structured DNA, such as insertion/ deletion loops, hairpins, and flap DNA [43,55]. Frameshift DNA mutations are mistakenly generated during replication of repetitive sequences [41], and as a result, the bulge DNA are produced by slipped misalignment of tandem repeats [56,57]. Rearrangements between tandem repeated DNA are important factors for genome instability and have been implicated in Friedreich ataxia in humans [58,59]. Slipped misalignment of tandem repeat DNA may cause palindrome-stimulated deletion or expansion by two RecA-independent recombination mechanisms-that is, singlestrand annealing and replication slipped mispairing [60,61]. Single-strand-specific exonucleases, such as ExoI, ExoX, and RecJ, were reported to stabilize tandem repeats and limit RecAindependent recombination [56,62]. However, the downstream structure-specific exonuclease of Endo V for the further trimming of the DNA from the broken end has not yet been identified.
Our structural and biochemical data of RNase T show that it can bind and digest bulge/bubble and Y-structured DNA. Moreover, RNase T can work with Endo V, DNA Polymerase I (Klenow fragment exo 2 ), and ligase to restore an inosine to cytosine in a heteroduplex DNA molecule in vitro. The crystal structures of RNase T bound with a blunt-end bulge DNA further show how RNase T removes the last base pair at the 39 end by a special Phe-inserting binding mode. All these results suggest that RNase T may function as the downstream exonuclease of Endo V in alternative DNA repair. Taking together these lines of evidence, we suggest that RNase T likely recognizes these bulge and bubble DNA structures generated by Endo V and trims at the 39 end of the nicked site to remove the last base pair next to the lesion. The single-stranded DNA or damaged DNA is then released for the next step of processing (see Figure 7A).
After removing the 39-end base-paired nucleotide by RNase T, insertion DNA, hairpin DNA, and deaminated DNA lesions are Figure 6. The DNA substrate preference comparison between RNase T, ExoI, and ExoX. (A) The 59-end 32 P-labelled single-stranded 11nucleotide DNA molecules were digested with similar efficiencies by RNase T, ExoI, and ExoX. (B) RNase T is more specific than ExoX in digesting a Ystructured DNA and generated the end product with a 1-nucleotide overhang. ExoI did not digest a Y-structured DNA at low enzyme concentrations. (C) RNase T is more specific than ExoX at digesting a duplex DNA with a short overhang to generate a more specific end product of a duplex with a 1nucleotide overhang. ExoI did not digest duplex DNA with a short overhang at low enzyme concentrations. doi:10.1371/journal.pbio.1001803.g006 released as single-stranded DNA. These single-stranded insertion DNA and hairpin DNA are probably further trimmed by the single-strand-specific exonucleases, such as ExoI and/or ExoX, with the help of single strand binding protein (SSB) and helicases. RNase T can further digest the 39-end short overhang close to the duplex region in a way that we observed in the crystal structure of the Y-structured DNA complex. Deaminated DNA lesions are likely also removed by RNase T since we show that RNase T can digest single-stranded DNA containing oxidized bases and deaminated bases ( Figure 1C). It has been shown that the dimeric Exo I from Thermus thermophilus shares a sequence homology to RNase T and plays a similar role in digesting damaged DNA with methylated and deaminated bases [30]. It is very likely that Exo I from Thermus thermophilus is a functional homologue of RNase T and both of them play key roles in DNA repair. Therefore, after nicking by Endo V, the single-strand-specific exonucleases and Figure 7. Possible roles of RNase T in Endo Vdependent, mismatch, and UV-induced DNA repair. Endo V makes a nick at the 39 side one base pair away from the damaged sites, including insertion loops and deaminated sites. RNase T further trims the bulge and bubble DNA and removes the last base-paired structure to release the single-stranded DNA insertions or damaged DNA base for the next-step processing. ExoI, ExoX, and RNase T further trim the flapped 39 overhang, followed by DNA polymerase and ligase activity to complete the repair pathway. (B) In the mismatch DNA repair pathway, a Y-structured DNA is produced after processing by MutS, MutL, and MutH. It is likely that ExoI and ExoX trim the long 39 tail with the help of SSB and RNase T is responsible for the final trimming of the short 39 overhang of the Y-structured DNA. (C) In the UV-induced DNA repair by replication restart pathway, the damaged DNA are bound and annealed by RecA and RecFOR. As a result, the Y-structured DNA (in the orange box) is generated that can be further digested by RNase T. (D) The Y-structured DNA can be generated in gap-filling homologous recombination and (E) RecA-dependent homologous recombination. RNase T may remove the short 39 overhang of these Y-structured DNA in these UV-induced DNA repair pathways. doi:10.1371/journal.pbio.1001803.g007 structure-specific RNase T likely work together to further trim DNA from the broken end. After this trimming, polymerases and ligases can complete the DNA repair pathway.

RNase T Digests Y-Structured DNA in Various DNA Repair Pathways
RNase T plays crucial roles in various DNA repair pathways, as shown by the sensitivity of the rnt knockout strain to a wide range of DNA-damaging agents. The indispensable role of RNase T might be due to its unique specificity for structured DNA that are generated during various DNA repair pathways. For instance, UV radiation can lead to single/double-strand breaks and base modifications, such as cross-linked pyrimidine dimers, photoproducts, and thymine glycols, and as a result, three different UV-induced DNA repair pathways are initiated [39,63]. In the first pathway, the base modification induced by UV may stall replication forks. In such a case, RecFOR and RecA bind to the lagging strand template and the invasion-containing leading strand to promote double-strand formation and repair by NER [64]. During this process, the Y-structured DNA formed on the leading strand requires a structure-specific exonuclease, very likely RNase T, to trim its 39 overhang (see Figure 7C).
In the second pathway, UV radiation can induce single-strand breaks that can be repaired by homologous recombination [65]. During this process, Y-structured DNA is formed as an intermediate during gap-filling recombination (see Figure 7D). ExoI was reported to promote this RecA-dependent 59-end strand exchange by digesting the 39 competitor strand [66,67]. However, ExoI cannot digest the 39 overhang close to the duplex region, and thus most likely RNase T is responsible for processing the Ystructured DNA intermediates in the gap-filling recombination pathway.
In the third pathway, the double-strand breaks induced by UV radiation are generally repaired by the RecA-dependent homologous recombination in bacteria [68]. This DNA repair pathway is initiated by RecBCD or RecJ to generate 39 overhangs and is followed by RecA and RecFOR to promote strand invasion. DNA repair synthesis is then primed by PolI and PolIII from the invaded strand of the D-loop structure. RuvC resolvase cleaves the Holliday junctions that are synthesized after branch migration and LigA seals the nick to complete the homologous recombination [1]. In this process, ExoI was reported to affect RecBCDmediated recombination [69] since the 39-59 exonucleases are required to degrade the 39 tail of the intermediate Y-structured DNA after RecA dissociation [48,51,70,71]. Yet ExoI is not an appropriate exonuclease for digesting the 39 tail near the duplex region. Based on our results, we suggest that most likely RNase T is involved in digesting the 39 tail close to the duplex region in the UV-induced DNA homologous recombination ( Figure 7E). Moreover, in comparison with FEN1, which is a flap endonuclease that binds DNA with one 39-flap nucleotide and cleaves one nucleotide into the double-stranded DNA at the 59 flap end to produce a ligatable product during DNA replication and repair [72], RNase T is likely required to produce a DNA with a short 39 overhang with one or two nucleotides that can be further processed in DNA homologous recombination.
Besides UV-induced DNA repair, RNase T may also participate in other DNA repair processes that require a structure-specific 39-59 exonuclease, such as MMR. It has been shown that ExoI and ExoX are essential for methyl-directed mismatch repair in E. coli [7,[10][11][12]49,50]. These two monomeric exonucleases are responsible for removing the 39 single-stranded tail in Y-structured DNA during MMR (see Figure 7B). However, they cannot process the 39 single-stranded tail close to the double-stranded region [1,49].
ExoI only processes DNA with a long single-stranded region (over 13 nucleotides) in a processive manner, while a SSB stimulates its exonuclease activity [52,73,74]. ExoX, however, interacts with MutL during MMR and is not specific for processing Y-structured DNA [8,12]. On the other hand, the RNase T homolog Thermus thermophilus ExoI is suggested to excise the 39 overhang of a Ystructured DNA and plays a role in MMR [30]. Therefore, it is very likely that RNase T processes the 39 tail of the Y-structured DNA in MMR in E. coli. Our structure and biochemical assays show that the C effect does not occur when RNase T digests short 39 overhang of a Y-structured DNA, and hence RNase T is capable of processing any sequence of the 39 overhang of a Ystructured DNA during MMR. Therefore, we propose here that the monomeric ExoI and ExoX work with a helicase or SSB to process long 39 tails, while the dimeric RNase T further trims the short 39 overhang of Y-structured DNA during MMR.
In conclusion, RNase T is a unique structure-specific exonuclease responsible for processing the 39 ends of structured DNA in various DNA repair pathways. RNase T has an ideal dimeric architecture for binding and processing the 39 end of various structured DNA in diverse ways, including duplex, bulge/bubble, and Y-structured DNA. Therefore, this intriguing exonuclease has multiple functions not only for processing duplex RNA during RNA maturation, but also processing bubble/bulge and Ystructured DNA during DNA repair. The diverse functions and different specificities of RNase T are closely correlated to its dimerization architecture and various binding modes against different substrates. We provide solid data here showing how the dimeric RNase T processes structured DNA in DNA repair that will serve as a model for understanding the molecular functions of thousands of members of DnaQ-like exonucleases.

Bacteria Strains and Survival Studies
Wild-type E. coli K-12, single gene knockout (Drnt, Dsbcb, Dexox, Dpnp, and DrecJ) strains used in the survival studies were from the Keio collection [75]. All E. coli cells were grown to an OD 600 of 0.5-0.6 in LB medium at 37uC. To measure the acute sensitivity to hydrogen peroxide (H 2 O 2 ), cells were exposed to 0, 20, 40, and 80 mM H 2 O 2 for 20 min. After removing H 2 O 2 , cells were diluted 100-fold into 10 ml LB medium and further grown on a rotary shaker (200 r.p.m.) at 37uC for the measurement of A 600 (OD) at 60 min intervals. To measure the chronic sensitivity to H 2 O 2 , MMS, mitomycin (MMC), and 4NQO, serial dilutions of cells were spotted on plates containing indicated concentrations of the DNA-damaging agents and incubated overnight at 37uC. To measure the sensitivity against UV-C, serial dilutions of cells were spotted on plates and exposed to UV-C (254 nm) in 20 J/m 2 for 10 s by Hoefer UVC 500-Ultraviolet Crosslinker (Hoefer Inc.). After UV-C irradiation, cells were incubated overnight at 37uC.

Protein Expression and Purification
The full-length rnt, sbcb, and exox genes were amplified by PCR using E. coli genomic DNA from JM109 or K-12 strains and cloned into NdeI/XhoI sites of expression vectors pET-28a (Novagen) to generate the N-terminal His-tagged fused recombinant proteins. The expression plasmid was transformed into the E. coli BL21-CodonPlus(DE3)-RIPL strain (Stratagene) cultured in LB medium supplemented with 35 mg/ml kanamycin. Cells were grown to an OD 600 of 0.5-0.6 at 37uC and induced by 0.8 mM IPTG at 18uC for 18 h. The harvested cells were dissolved in 50 mM Tris-HCl (pH 7.5) buffer containing 300 mM NaCl and disrupted by a microfluidizer. Each exonuclease was purified by chromatographic methods using a HiTrap TALON column (GE Healthcare), a HiTrap Heparin column (GE Healthcare), and a gel filtration column (Superdex 75, GE Healthcare). Purified RNase T, ExoI, and ExoX samples were concentrated to 15-35 mg/ml in 300 mM NaCl and 50 mM Tris-HCl (pH 7.0).

DNA Digestion and Binding Assays
DNA oligonucleotides used for nuclease activity assays were synthesized (BEX Co., Tokyo, Japan or MDBio, Inc., Taiwan) and labeled at the 59 end with [c-32 P]ATP by T4 polynucleotide kinase and purified on a Microspin G-25 column (GE Healthcare) to remove the nonincorporated nucleotides. Purified substrates (20 nM; see Table S1 for sequences) were incubated with RNase T, ExoI, or ExoX at various concentrations in a buffered solution of 120 mM NaCl, 2 mM MgCl 2 , and 50 mM Tris-HCl (pH 7.0) at room temperature for 20-60 min. The reaction was quenched by addition of the stop solution (26 TBE) and heating at 95uC for 5 min. Reaction samples were then resolved on 20% denaturing polyacrylamide gels and visualized by autoradiography (Fujifilm, FLA-5000).
DNA binding affinities of RNase T, ExoI, and ExoX were measured by gel shift assays. The 59-end 32 P-labeled DNA substrates (20 nM) were incubated with RNase T, ExoI, or ExoX in a solution of 100 mM NaCl, 30 mM EDTA, 10 mM EGTA, and 50 mM Tris-HCl (pH 7.0) for 20 min at room temperature. The concentrations of each protein used in the assays were 0, 5, and 50 mM. Reaction samples were then resolved on 20% TBE gels (Invitrogen) and visualized by autoradiography (Fujifilm, FLA-5000).

In Vitro Endo V-Dependent DNA Repair Assay
The E. coli strain NM522.RS5033 was used in the assay as described in Fang et al. [76]. DNA polymerase I (E. coli), the Klenow fragment exo 2 (DNA polymerase I Klenow fragment without the 39-59 exonuclease activity), E. coli DNA ligase, T4 polynucleotide kinase, recombinant Endo V, and restriction endonucleases were obtained from New England Biolabs. RecBCD nuclease was purchased from EPICENTRE Biotechnologies.
Construction of dI-G heteroduplex DNA substrates was prepared as described in Lee et al. [46]. M13mp18 replicative form DNA was hydrolyzed with HindIII and mixed with a 4-fold molar excess of M13LR1 viral DNA, followed by alkaline denaturation and re-annealing. The excess ssDNA was removed by hydroxyapatite (Biorad) chromatography and benzoylated naphthylated DEAE cellulose (Sigma) chromatography, and the linear dsDNA was removed by RecBCD nuclease (EPICENTRE) treatment. The resulting circular duplex DNA containing 22-nt gaps was further purified by Vivaspin 20 ultrafiltration (GE Healthcare). A 59-phosphorylated deoxyinosine-containing 22-bp synthetic oligonucleotides, 59-AGCTCTIGAGGCTGCTGCT-GCT-39 (Blossom Biotech), was then annealed to the gap and sealed by T4 DNA ligase in the presence of ethidium bromide. The covalently closed dG:I heteroduplex DNA was isolated by CsCl-EtBr density gradient centrifugation.
The repair conditions were modified from Lee et al. [46]. The dI-G heteroduplex substrates (0.1 mg) were incubated with repair enzymes (1.1 nM Endo V, 0.13 mM DNA polymerase I/0.13 mM Klenow fragment exo-, and 5 mM RNaseT) for 30 min at 37uC in 15-ml reactions containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM dithiothreitol, 50 mg/ml bovine serum albumin, 0.3 mM NAD + , and 125 mM of each dNTP. The reactions were terminated by heat inactivation at 75uC for 20 min. The DNA was then analyzed by restriction endonuclease hydrolysis and agarose gel electrophoresis. The gel images were captured by a gel documentation CCD camera (UVP Ltd.) using Viewfinder 3.0, and band intensities were then measured by NIH Image J 1.45s software.

Crystallization and Crystal Structure Determination
Wild-type RNase T (25-35 mg/ml) in 300 mM NaCl and 50 mM Tris-HCl (pH 7.0) were mixed with different stem-loop DNA substrates in the molar ratio of 1:1.2. Detailed information for DNA sequences and crystallization conditions of the three structures is given in the Table S2. All crystals were cryo-protected by Paraton-N (Hampton Research, USA) for the data collection at 100 K. X-ray diffraction data were collected using synchrotron radiations at SPXF beamline BL13B1 at NSRRC, Taiwan, or at the BL44XU beamline at SPring-8, Japan. All diffraction data were processed by HKL2000, and the diffraction statistics are listed in Table 1. Structures were solved by the molecular replacement method using the crystal structure of E. coli RNase T (PDB ID code 3NGY) as the search model by program MOLREP of CCP4. The models were built by Coot and refined by Phenix.

Accession Numbers
Structural coordinates and diffraction structure factors have been deposited in the RCSB Protein Data Bank with the PDB ID codes of 4KB0 and 4KB1 for RNase T-bulge DNA complexes and 4KAZ for the RNase T-Y structured-DNA complex. Figure S1 Effects of UV and DNA-damaging agents on various exonuclease-deficient E. coli K-12 strains. (A) The ExoI, ExoX, PNPase, and RecJ knockout cells were exposed to UV-C for 10 s or different DNA-damaging agents, such as H 2 O 2 , MMS, 4NQO, and MMC, in a chronic dose. The rnt rescue experiments for RNase T knockout cells were performed in parallel by transforming the rnt-containing plasmid into RNase T knockout cells, which were then exposed to UV-C for 10 s (Drntrnt). The rnt rescued the sensitivity of the RNase T knockout cells against UV-C. (B) Growth curves of the exonuclease-deficient cells after exposure to H 2 O 2 in an acute does. The rnt rescue experiments (Drnt-rnt) were performed in parallel showing that rnt rescued the sensitivity of the RNase T knockout cells against H 2 O 2 . The rnt-containing plasmid was prepared as described previously [27]. (TIF) Substrates for these experiments were single-stranded 11-nucleotide DNA (ssDNA 11) and stem-loop DNA with 0-, 4-, 6-, and 10nucleotide 39 overhang (SL_0, SL_4, SL_6, and SL_100). Sequences of these DNAs are listed in Table S1. (TIF) Movie S1 How RNase T processes duplex DNA with a 39 overhang.