Showing papers by "Graham C. Walker published in 2006"

DNA Repair and Mutagenesis

Abstract: DNA damage Mutations The reversal of base damage Base excision repair Nucleotide excision repair in prokaryotes Nucleotide excision repair in lower eukaryotes Nucleotide excision repair in mammalian cells: general considerations and chromatin dynamics Nucleotide excision repair in mammalian cells: genes and proteins Mismatch repair The SOS response and recombinational repair in prokaryotes Mutagenesis in prokaryotes Mutagenesis in eukaryotes Other DNA damage tolerance responses in eukaryotes Hereditary diseases with defective responses to DNA damage

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Abstract: Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance1. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD′2C), respectively2,3. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic4. 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 κ, 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.

The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G2/M phase rather than S phase

Abstract: The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase ζ (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase ζ are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein–protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are ≈50-fold higher in G2 and throughout mitosis than during G1 and much of S phase. Differential survival of a rev1Δ strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.

Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12

Abstract: An insight into a previously unknown step in B12 biosynthesis was unexpectedly obtained through our analysis of a mutant of the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. This mutant was identified based on its unusually bright fluorescence on plates containing the succinoglycan binding dye calcofluor. The mutant contains a Tn5 insertion in a gene that has not been characterized previously in S. meliloti. The closest known homolog is the bluB gene of Rhodobacter capsulatus, which is implicated in the biosynthesis of B12 (cobalamin). The S. meliloti bluB mutant is unable to grow in minimal media and fails to establish a symbiosis with alfalfa, and these defects can be rescued by the addition of vitamin B12 (cyanocobalamin) or the lower ligand of cobalamin, 5,6-dimethylbenzimidazole (DMB). Biochemical analysis demonstrated that the bluB mutant does not produce cobalamin unless DMB is supplied. Sequence comparison suggests that BluB is a member of the NADH/flavin mononucleotide (FMN)-dependent nitroreductase family, and we propose that it is involved in the conversion of FMN to DMB.

Y‐family DNA polymerases respond to DNA damage‐independent inhibition of replication fork progression

Abstract: In Escherichia coli, the Y-family DNA polymerases Pol IV (DinB) and Pol V (UmuD2′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 umuC122∷Tn5 allele of umuC, dinB, and certain forms of umuD gene products endow E. coli with the ability to withstand HU treatment (HUR). The catalytic activities of the UmuC122 and DinB proteins are both required for HUR. 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.

Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes

Abstract: Legumes establish mutualistic associations with nitrogen-fixing bacteria, commonly referred to as rhizobia. Most rhizobia enter roots of host plants via root hairs, where they induce the formation of infection threads. Bacterial invasion and formation of nodules containing nitrogen-fixing bacteroids (the Fix+ phenotype) depend on a molecular dialogue between the symbiotic partners. Host plants secrete flavonoids that induce the synthesis of rhizobial lipo-chito-oligosaccharides called Nod factors (24, 26) and perceive them via a specific signal transduction pathway that culminates in expression of symbiosis-specific plant genes required for nodule development (34). In many symbiotic associations, nodule formation also depends on rhizobial macromolecules that include exopolysaccharides (EPS), lipopolysaccharides, K-antigens, and cyclic β-glucans. These extracellular polysaccharides (or the oligosaccharides derived from them) are crucial in the early stages of nodule formation that begins after rhizobia have entered the host plant (3, 8, 17, 19, 25, 33, 36). In addition to a protective role, rhizobial EPS have vital but poorly understood roles in infection of legume roots (2). The EPS structures of various rhizobia, including Rhizobium sp. strain NGR234 (also called Sinorhizobium fredii NGR234), which nodulates more than 112 genera of legumes (30), have been described. The repeating subunit of the acidic EPS of NGR234 is a nonasaccharide consisting of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues (9) (Fig. ​(Fig.1A).1A). Mutants of NGR234 deficient in EPS synthesis formed nonmucoid (“dry”) colonies on agar plates and lost the capacity to induce the formation of nitrogen-fixing nodules on Leucaena leucocephala (Fix− phenotype). Instead, small empty nodules (pseudonodules) were formed (5). A number of genes of NGR234 involved in synthesis of EPS have been identified in an exo cluster (6, 15, 45), which has been sequenced (38) (Fig. ​(Fig.1B).1B). This cluster is located on the pNGR234b megaplasmid and is similar to the exo cluster of Sinorhizobium meliloti strain 1021 that is required for synthesis of succinoglycan (also called EPS I) (13). Based on known or assumed functions of exo genes in S. meliloti (2, 32), we predict that EPS synthesis in NGR234 depends on the glycosyl transferases (ExoY, ExoA, ExoL, ExoM, ExoO, and ExoU) that form an undecaprenol diphosphate-linked repeating subunit at the cytoplasmic face of the inner membrane. Probably, the lipid-linked subunits are then flipped across the inner membrane and finally assembled by an ExoQ/ExoP/ExoF-dependent polymerization system that is similar to the Wzy polymerization pathway of group 1 capsular polysaccharides in Escherichia coli (41). In this model, ExoQ is a putative polymerase and ExoP is a copolymerase belonging to the MPA1 (cytoplasmic membrane-periplasmic auxiliary protein 1) family. ExoF is probably an outer membrane auxiliary protein required for EPS export. Related genes required for secretion and export of EPS (pssL, pssT, pssP, and pssN) have been identified in Rhizobium leguminosarum bv. trifolii (reference 22 and references therein). FIG. 1. (A) Repeating subunit of the acidic EPS from NGR234 (9). Additional acetyl groups are present at unidentified positions. (B) Genetic map of the exo cluster of pNGR234b (38). The exs genes and neighboring exoB gene are not shown. BamHI fragments were cloned ... Low-molecular-weight (LMW) forms of EPS (called exo-oligosaccharides [EOSs]) have been identified in supernatants of rhizobial cultures. Nodulation experiments with purified LMW EPS applied to roots indicated that EOSs could complement the symbiotic defects of exo mutants under certain growth conditions (1, 10, 14, 40). Similar complementation effects were observed when exo mutants were coinoculated with noninvasive strains that synthesize EPS (17, 18). Although not always conclusive, the results of such complementation experiments suggest that EOSs are symbiotically active molecules in certain plants. Synthesis of EOSs seems to be controlled either by enzymes involved in export and polymerization of the lipid-linked repeating EPS subunit or by extracellular glycanases that release EOSs from high-molecular-weight (HMW) forms (14). Glycanases that cleave EPS have been identified in S. meliloti and R. leguminosarum bv. viciae. Corresponding mutants produced lower levels of EOSs without altering the symbiotic phenotype (12, 43). The previously characterized glycanase ExoK secreted by S. meliloti (43, 44) exhibits a high level of sequence similarity [e−129] to the putative ExoK protein of NGR234. In this study, we mutated exo genes of NGR234 involved in synthesis of the repeating subunit (exoL, exoY, and exoZ), polymerization (exoF, exoP, and exoQ), and production of EOSs (exoK). We provide evidence that EOSs of NGR234 are required for symbiosis with certain plants.

Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors

Abstract: Members of the Y family of DNA polymerases are specialized to replicate lesion-containing DNA However, they lack 3'-5' exonuclease activity and have reduced fidelity compared to replicative polymerases when copying undamaged templates, and thus are potentially mutagenic Y family polymerases must be tightly regulated to prevent aberrant mutations on undamaged DNA while permitting replication only under conditions of DNA damage These polymerases provide a mechanism of DNA damage tolerance, confer cellular resistance to a variety of DNA-damaging agents, and have been implicated in bacterial persistence The Y family polymerases are represented in all domains of life Escherichia coli possesses two members of the Y family, DNA pol IV (DinB) and DNA pol V (UmuD'(2)C), and several regulatory factors, including those encoded by the umuD gene that influence the activity of UmuC This chapter outlines procedures for in vivo and in vitro analysis of these proteins Study of the E coli Y family polymerases and their accessory factors is important for understanding the broad principles of DNA damage tolerance and mechanisms of mutagenesis throughout evolution Furthermore, study of these enzymes and their role in stress-induced mutagenesis may also give insight into a variety of phenomena, including the growing problem of bacterial antibiotic resistance

Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions.

Abstract: In all eukaryotes examined to date, Rev1 and the REV3/REV7-encoded polymerase zeta (Polζ) are absolutely required for the mutagenic bypass of DNA lesions, a process called translesion DNA synthesis (TLS). Even on undamaged DNA, most TLS occurs at a low fidelity, relative to replicative DNA synthesis, and represents a major source of DNA damage-induced base substitutions and frameshift mutations (7, 36). In the yeast Saccharomyces cerevisiae, the majority of mutagenic DNA lesion bypass is carried out by Polζ, a member of the B family of DNA polymerases, and the Y-family polymerase Rev1 (21). Both Polζ and Rev1 are part of the “error-prone” branch of the RAD6 epistasis pathway (20). Inactivating mutations in the rev genes render cells sensitive to DNA-damaging agents and confer a “reversionless” phenotype after DNA damage (23, 25). Together, Polζ and Rev1 functions are required for the generation of 95% to 98% of UV radiation (UV)-induced base pair substitutions (22, 24). Though the yeast Polζ can, in some cases, insert nucleotides across damaged bases, it possesses a unique facility to extend DNA synthesis from terminally mismatched primers and, in general, from distorted DNA structures (15, 45). The Polζ enzyme consists of a catalytic subunit, Rev3, and an accessory subunit, Rev7, that enhances the polymerase activity of Rev3 by about 200-fold (33). Polζ replicates past abasic sites in the DNA template, an activity that is greatly stimulated by Rev1 in vitro (32). The Rev1 polymerase is endowed with the unique ability to insert predominantly dCMP both across an undamaged template G and abasic sites as well as a variety of other DNA lesions (11, 21, 32). In this capacity, Rev1 uses a novel mechanism of DNA synthesis whereby the incoming dCTP pairs with an arginine rather than the template base, and the template G is evicted from the DNA helix (30). Intriguingly, the catalytic activity of Rev1 appears not to be essential for the bypass of DNA damage, as mutations inactivating this activity leave Rev1 still competent to participate in translesion synthesis, (12; L. Waters, S. D'Souza and G. C. Walker, unpublished observations), albeit with an altered mutation spectrum (35). In contrast to the polymerase domain, the N-terminal BRCT motif of yeast Rev1 is required for mutagenesis. A G193R point mutation in the Rev1 BRCT motif (the rev1-1 allele) abrogates the ability of Rev1 to function in induced mutagenesis and confers a moderate sensitivity to DNA damaging agents (19, 25). BRCT domains (BRCA1 C terminus) are ubiquitous motifs that facilitate physical interactions among proteins involved in the cellular response to DNA damage (4, 44). Interestingly, the Rev1-1 protein retains its catalytic dCMP transferase activity in vitro (31). Thus, the TLS function of yeast Rev1 is thought to require protein-protein interactions, rather than its polymerase activity, and has been proposed to nucleate a protein complex at the site of a stalled replication fork, possibly to facilitate a switch between the replicative DNA polymerase and other TLS polymerases (12). However, the recent observation that the yeast Rev1 protein levels fluctuate dramatically in the yeast cell cycle and peak during the G2/M phase suggests that Rev1 functions mainly in the G2/M phase rather than during the S phase of the cell cycle when replication occurs (46). In addition to its BRCT and polymerase domains, Rev1 possesses a C-terminal region which, in higher eukaryotes, has been shown to interact with a variety of translesion DNA polymerases, including Polκ, Polλ, Polη, and Polι and the Polζ accessory subunit, Rev7 (9, 29, 34, 40, 41). Unlike human and mouse Rev1, relatively little is known about the C terminus of the Saccharomyces cerevisiae Rev1 protein. Due to the poor sequence conservation of the C termini between yeast Rev1 and its vertebrate homologs, it has been suggested that the C terminus of yeast Rev1 plays no part in the process of mutagenic bypass or protein-protein interactions (16, 29, 41). In this study, we assess the contribution of the C terminus of yeast Rev1 in DNA damage tolerance by overproducing a C-terminal fragment in cells exposed to DNA damage. We find that overproduction of the extreme C terminus of the yeast Rev1 protein confers a strong dominant-negative phenotype on the survival and reversion frequencies of otherwise wild-type cells exposed to DNA damage. We show that the dominant-negative phenotype of the Rev1 C terminus requires both REV1 and REV7, providing the first in vivo evidence for a critical function of the C terminus of Saccharomyces cerevisiae Rev1 in DNA damage tolerance. We use immunoprecipitation experiments to demonstrate that two novel regions of the Rev1 protein, the extreme C terminus and the BRCT region, independently interact with the Rev7 protein, the accessory subunit of Polζ. Consistent with an earlier observation (1) showing a direct interaction between the Rev1 polymerase-associated domain (PAD) and Rev7, we report that the Rev1 PAD also immunoprecipitates Rev7. Our results thus significantly expand the study of the interaction between Rev1 and Rev7 and have important implications for the process of translesion DNA synthesis.

CbrA Is a Stationary-Phase Regulator of Cell Surface Physiology and Legume Symbiosis in Sinorhizobium meliloti

Abstract: Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34

Abstract: Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B(12)-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.

Two processivity clamp interactions differentially alter the dual activities of UmuC

Abstract: DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.

A Non‐cleavable UmuD variant that acts as a UmuD’ mimic

Abstract: UmuD2 cleaves and removes its N-terminal 24 amino acids to form UmuD′2, which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the β processivity clamp (“β”) of the replicative DNA polymerase, pol III. A possible β-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with β. We performed alanine-scanning mutagenesis of this motif (14TFPLF18) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the β-binding motif variant bind to β with similar Kd values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in β is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD′, such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD′ mimic.

A small-scale concept-based laboratory component: the best of both worlds.

Abstract: In this article, we describe an exploratory study of a small-scale, concept-driven, voluntary laboratory component of Introductory Biology at the Massachusetts Institute of Technology. We wished to investigate whether students' attitudes toward biology and their understanding of basic biological principles would improve through concept-based learning in a laboratory environment. With these goals in mind, and using our Biology Concept Framework as a guide, we designed laboratory exercises to connect topics from the lecture portion of the course and highlight key concepts. We also strove to make abstract concepts tangible, encourage learning in nonlecture format, expose the students to scientific method in action, and convey the excitement of performing experiments. Our initial small-scale assessments indicate participation in the laboratory component, which featured both hands-on and minds-on components, improved student learning and retention of basic biological concepts. Further investigation will focus on improving the balance between the minds-on concept-based learning and the hands-on experimental component of the laboratory.

BacA-Mediated Bleomycin Sensitivity in Sinorhizobium meliloti Is Independent of the Unusual Lipid A Modification

Abstract: Sinorhizobium meliloti bacA mutants are symbiotically defective, deoxycholate sensitive, and bleomycin resistant. We show that the bleomycin resistance phenotype is independent of the lipid A alteration and that the changes giving rise to both phenotypes are likely to be involved in the inability of bacA mutants to persist within their hosts.