Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

Be they prokaryotic or eukaryotic, organisms are exposed to a multitude of deoxyribonucleic acid (DNA) damaging agents ranging from ultraviolet (UV) light to fungal metabolites, like Aflatoxin B1. Furthermore, DNA damaging agents, such as reactive oxygen species, can be produced by cells themselves as metabolic byproducts and intermediates. Together, these agents pose a constant threat to an organism's genome. As a result, organisms have evolved a number of vitally important mechanisms to repair DNA damage in a high fidelity manner. They have also evolved systems (cell cycle checkpoints) that delay the resumption of the cell cycle after DNA damage to allow more time for these accurate processes to occur. If a cell cannot repair DNA damage accurately, a mutagenic event may occur. Most bacteria, including Escherichia coli, have evolved a coordinated response to these challenges to the integrity of their genomes. In E. coli, this inducible system is termed the SOS response, and it controls both accurate and potentially mutagenic DNA repair functions [reviewed comprehensively in () and also in ()]. Recent advances have focused attention on the umuD(+)C(+)-dependent, translesion DNA synthesis (TLS) process that is responsible for SOS mutagenesis (). Here we discuss the SOS response of E. coli and concentrate in particular on the roles of the umuD(+)C(+) gene products in promoting cell survival after DNA damage via TLS and a primitive DNA damage checkpoint.

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.

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen that survives desiccation and quickly acquires resistance to multiple antibiotics. Escherichia coli gains antibiotic resistances by expressing genes involved in a global response to DNA damage. Therefore, we asked whether A. baumannii does the same through a yet undetermined DNA damage response akin to the E. coli paradigm. We found that recA and all of the multiple error-prone DNA polymerase V (Pol V) genes, those organized as umuDC operons and unlinked, are induced upon DNA damage in a RecA-mediated fashion. Consequently, we found that the frequency of rifampin-resistant (Rif r ) mutants is dramatically increased upon UV treatment, alkylation damage, and desiccation, also in a RecA-mediated manner. However, in the recA insertion knockout strain, in which we could measure the recA transcript, we found that recA was induced by DNA damage, while uvrA and one of the unlinked umuC genes were somewhat derepressed in the absence of DNA damage. Thus, the mechanism regulating the A. baumannii DNA damage response is likely different from that in E. coli. Notably, it appears that the number of DNA Pol V genes may directly contribute to desiccation-induced mutagenesis. Sequences of the rpoB gene from desiccation-induced Rif r mutants showed a signature that was consistent with E. coli DNA polymerase V-generated base-pair substitutions and that matched that of sequenced A. baumannii clinical Rif r isolates. These data strongly support an A. baumannii DNA damage-inducible response that directly contributes to antibiotic resistance acquisition, particularly in hospitals where A. baumannii desiccates and tenaciously survives on equipment and surfaces.

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We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.Many bacteria have the ability to release sialic acids from complex glycoproteins and oligosaccharides present in the media or on cell surfaces at sites of colonization or infection. To use the released sialic acids as a rich source of carbon and nitrogen for growth, bacteria must have the ability to transport these compounds into the cell and convert the nine carbon sugars into intermediates that enter the central glycolytic pathways. The utilization of N-acetyl neuraminic acid (NANA), one of the sialic acids, has been well studied in Escherichia coli (36, 37), Haemophilus spp. (1, 35), and Clostridium spp. (38), to name a few.In many microorganisms, the genes for NANA utilization are arranged in an operon that may be regulated by a repressor protein, termed NanR. A comprehensive review of the organization and composition of several prokaryotic operons involved in NANA utilization has been published (36). Many of these operons share common components, including a transport gene for NANA (nanT), a gene encoding an aldolase (nanA) that splits NANA into pyruvate and N-acetyl mannosamine (manNAc), a gene encoding a kinase activity (nanK) that phosphorylates manNAc to form manNAc 6-P and, finally, an epimerase gene (nanE) whose product converts manNAc 6-P to N-acetylglucosamine 6-P (NAG 6-P). NAG 6-P then enters the common pathway of aminosugar utilization (21). For a schematic of the NANA utilization pathway in E. coli, the current paradigm of prokaryotic NANA utilization, see Fig. 7A.Bacteroides fragilis possesses a neuraminidase activity, which can liberate free NANA from complex glycoproteins and oligosaccharides. Go...

In Escherichia coli, the Y-family DNA polymerases Pol IV (DinB) and Pol V (UmuD 2 0 C) enhance cell survival upon DNA damage by bypassing replication-blocking DNA lesions. We report a unique function for these polymerases when DNA replication fork progression is arrested not by exogenous DNA damage, but with hydroxyurea (HU), thereby inhibiting ribonucleotide reductase, and bringing about damage-independent DNA replication stalling. Remarkably, the umuC122HTn5 allele of umuC, dinB, and certain forms of umuD gene products endow E. coli with the ability to withstand HU treatment (HU R ). The catalytic activities of the UmuC122 and DinB proteins are both required for HU R . Moreover, the lethality brought about by such stalled replication forks in the wild-type derivatives appears to proceed through the toxin/antitoxin pairs mazEF and relBE. This novel function reveals a role for Y-family polymerases in enhancing cell survival under conditions of nucleotide starvation, in addition to their established functions in response to DNA damage.

NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.