Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

A Single Residue Unique to DinB-Like Proteins Limits Formation of the Polymerase IV Multiprotein Complex in Escherichia coli

The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

Characterization of three mycobacterial DinB (DNA polymerase IV) paralogs highlights DinB2 as naturally adept at ribonucleotide incorporation

This study unveils Mycobacterium smegmatis DinB2 as the founder of a clade of Y-family DNA polymerase that is naturally adept at incorporating ribonucleotides by virtue of a leucine in lieu of a canonical aromatic steric gate. DinB2 efficiently scavenges limiting dNTP and rNTP substrates in the presence of manganese. DinB2''s sugar selectivity factor, gauged by rates of manganese-dependent dNMP versus rNMP addition, is 2.7- to 3.8-fold. DinB2 embeds ribonucleotides during DNA synthesis when rCTP and dCTP are at equimolar concentration. DinB2 can incorporate at least 16 consecutive ribonucleotides. In magnesium, DinB2 has a 26- to 78-fold lower affinity for rNTPs than dNTPs, but only a 2.6- to 6-fold differential in rates of deoxy versus ribo addition (k_pol). Two other M. smegmatis Y-family polymerases, DinB1 and DinB3, are characterized here as template-dependent DNA polymerases that discriminate strongly against ribonucleotides, a property that, in the case of DinB1, correlates with its aromatic steric gate side chain. We speculate that the unique ability of DinB2 to utilize rNTPs might allow for DNA repair with a ‘ribo patch’ when dNTPs are limiting. Phylogenetic analysis reveals DinB2-like polymerases, with leucine, isoleucine or valine steric gates, in many taxa of the phylum Actinobacteria.     

Roles of E. coli double-strand-break-repair proteins in stress-induced mutation

Special mechanisms of mutation are induced during growth-limiting stress and can generate adaptive mutations that permit growth. These mechanisms may provide improved models for mutagenesis in antibiotic resistance, evolution of pathogens, cancer progression and chemotherapy resistance. Stress-induced reversion of an Escherichia coli episomal lac frameshift allele specifically requires DNA double-strand-break-repair (DSBR) proteins, the SOS DNA-damage response and its error-prone DNA polymerase, DinB. We distinguished two possible roles for the DSBR proteins. Each might act solely upstream of SOS, to create single-strand DNA that induces SOS. This could upregulate DinB and enhance mutation globally. Or any or all of them might function other than or in addition to SOS promotion, for example, directly in error-prone DSBR. We report that in cells with SOS genes derepressed constitutively, RecA, RuvA, RuvB, RuvC, RecF, and TraI remain required for stress-induced mutation, demonstrating that these proteins act other than via SOS induction. RecA and TraI also act by promoting SOS. These and additional results with hyper-mutating recD and recG mutants support roles for these proteins via error-prone DSBR. Such mechanisms could localize stress-induced mutagenesis to small genomic regions, a potentially important strategy for adaptive evolution, both for reducing additional deleterious mutations in rare adaptive mutants and for concerted evolution of genes.     

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.     

Mycobacterium smegmatis DinB2 misincorporates deoxyribonucleotides and ribonucleotides during templated synthesis and lesion bypass

Mycobacterium smegmatis DinB2 is the founder of a clade of Y-family DNA polymerase that is naturally adept at utilizing rNTPs or dNTPs as substrates. Here we investigate the fidelity and lesion bypass capacity of DinB2. We report that DinB2 is an unfaithful DNA and RNA polymerase with a distinctive signature for misincorporation of dNMPs, rNMPs and oxoguanine nucleotides during templated synthesis in vitro. DinB2 has a broader mutagenic spectrum with manganese than magnesium, though low ratios of manganese to magnesium suffice to switch DinB2 to its more mutagenic mode. DinB2 discrimination against incorrect dNTPs in magnesium is primarily at the level of substrate binding affinity, rather than k_pol. DinB2 can incorporate any dNMP or rNMP opposite oxo-dG in the template strand with manganese as cofactor, with a kinetic preference for synthesis of an A:oxo-dG Hoogsteen pair. With magnesium, DinB2 is adept at synthesizing A:oxo-dG or C:oxo-dG pairs. DinB2 effectively incorporates deoxyribonucleotides, but not ribonucleotides, opposite an abasic site, with kinetic preference for dATP as the substrate. We speculate that DinB2 might contribute to mycobacterial mutagenesis, oxidative stress and quiescence, and discuss the genetic challenges to linking the polymerase biochemistry to an in vivo phenotype.     

An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents

DinB (DNA Pol IV) is a translesion (TLS) DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2)-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ), a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS). Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.     

UmuD2 Inhibits a Non-covalent Step during DinB-mediated Template Slippage on Homopolymeric Nucleotide Runs

Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating −1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD₂. In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD₂. Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD₂. We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD₂, when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.     

The DinB superfamily includes novel mycothiol, bacillithiol, and glutathione S-transferases

The superfamily of glutathione S-transferases has been the subject of extensive study; however, Actinobacteria produce mycothiol (MSH) in place of glutathione, and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates, an MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning, and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily, but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887, but En. faecalis produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Because more than two-thirds of the sequences assigned to the DinB superfamily are members of these families, it seems likely that such activity is dominant in the DinB superfamily.     

UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB

DinB is the only translesion Y family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage-inducible proteins UmuD(2) and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins, thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD(2)-interacting surface on DinB statistically covary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD(2) may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

Despite nearly universal conservation through evolution, the precise function of the DinB/pol κ branch of the Y-family of DNA polymerases has remained unclear. Recent results suggest that DinB orthologs from all domains of life proficiently bypass replication blocking lesions that may be recalcitrant to DNA repair mechanisms. Like other translesion DNA polymerases, the error frequency of DinB and its orthologs is higher than the DNA polymerases that replicate the majority of the genome. However, recent results suggest that some Y-family polymerases, including DinB and pol κ, bypass certain types of DNA damage with greater proficiency than an undamaged template. Moreover, they do so relatively accurately. The ability to employ this mechanism to manage DNA damage may be especially important for types of DNA modification that elude repair mechanisms. For these lesions, translesion synthesis may represent a more important line of defense than for other types of DNA damage that are more easily dealt with by other more accurate mechanisms.     

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

Background

Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins.

Methods

We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays.

Results

BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity.

Conclusions

BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules.

General significance

BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.     

Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain.

The UmuC/DinB family of bypass polymerases is responsible for translesion DNA synthesis and includes the human polymerases eta, iota, and kappa. We determined the 2.3 A resolution crystal structure of a catalytic fragment of the DinB homolog (Dbh) polymerase from Sulfolobus solfataricus and show that it is nonprocessive and can bypass an abasic site. The structure of the catalytic domain is nearly identical to those of most other polymerase families. Homology modeling suggests that there is minimal contact between protein and DNA, that the nascent base pair binding pocket is quite accessible, and that the enzyme is already in a closed conformation characteristic of ternary polymerase complexes. These observations afford insights into the sources of low fidelity and low processivity of the UmuC/DinB polymerases.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Efficient extension of slipped DNA intermediates by DinB is required to escape primer template realignment by DnaQ

We show that Escherichia coli DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the -4 position. A DinB(Y79L) variant, which extends these substrates less efficiently in vitro, allows the proofreading function of polymerase III to reverse their formation in vivo.     

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2′-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.     

DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate

DNA polymerase kappa (Polκ) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Polκ from the protozoan Trypanosoma cruzi (TcPolκ). The role of this TcPolκ copy was investigated by analysing its subcellular localization, its activities in vitro , and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C₂HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPolκ efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPolκ increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.