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1.
PLoS Biol ; 21(4): e3002085, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37079643

RESUMEN

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker's yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels of exo1 DNA-binding mutants to levels that approached the exo1 null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Intercambio Genético , Roturas del ADN de Cadena Simple , ADN Cruciforme , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Meiosis/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
J Bacteriol ; 204(4): e0061121, 2022 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-35285726

RESUMEN

Translesion synthesis (TLS) by specialized DNA polymerases (Pols) is an evolutionarily conserved mechanism for tolerating replication-blocking DNA lesions. Using the Escherichia coli dinB-encoded Pol IV as a model to understand how TLS is coordinated with the actions of the high-fidelity Pol III replicase, we previously described a novel Pol IV mutant containing a threonine 120-to-proline mutation (Pol IV-T120P) that failed to exchange places with Pol III at the replication fork in vitro as part of a Pol III-Pol IV switch. This in vitro defect correlated with the inability of Pol IV-T120P to support TLS in vivo, suggesting Pol IV gains access to the DNA, at least in part, via a Pol III-Pol IV switch. Interaction of Pol IV with the ß sliding clamp and the single-stranded DNA binding protein (SSB) significantly stimulates Pol IV replication and facilitates its access to the DNA. In this work, we demonstrate that Pol IV interacts physically with Pol III. We further show that Pol IV-T120P interacts normally with the ß clamp, but is impaired in interactions with the α catalytic and εθ proofreading subunits of Pol III, as well as SSB. Taken together with published work, these results provide strong support for the model in which Pol IV-Pol III and Pol IV-SSB interactions help to regulate the access of Pol IV to the DNA. Finally, we describe several additional E. coli Pol-Pol interactions, suggesting Pol-Pol interactions play fundamental roles in coordinating bacterial DNA replication, DNA repair, and TLS. IMPORTANCE Specialized DNA polymerases (Pols) capable of catalyzing translesion synthesis (TLS) generate mutations that contribute to bacterial virulence, pathoadaptation, and antimicrobial resistance. One mechanism by which the bacterial TLS Pol IV gains access to the DNA to generate mutations is by exchanging places with the bacterial Pol III replicase via a Pol III-Pol IV switch. Here, we describe multiple Pol III-Pol IV interactions and discuss evidence that these interactions are required for the Pol III-Pol IV switch. Furthermore, we describe several additional E. coli Pol-Pol interactions that may play fundamental roles in managing the actions of the different bacterial Pols in DNA replication, DNA repair, and TLS.


Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , ADN/metabolismo , ADN Polimerasa III/genética , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
3.
J Bacteriol ; 203(23): e0030321, 2021 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-34543108

RESUMEN

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess ß clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the ßE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, ßE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant ßE202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the ß clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The ß clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the ß clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant ß clamps that fail to inhibit growth. Their analysis revealed that ßE202K is unique among them. Our work offers new insights into how the ß clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.


Asunto(s)
ADN Polimerasa III/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Sustitución de Aminoácidos , ADN Polimerasa III/genética , Escherichia coli/genética , Modelos Moleculares , Mutación , Conformación Proteica
4.
J Bacteriol ; 203(23): e0030421, 2021 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-34543109

RESUMEN

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (ßE202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaNE202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the ß clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaNE202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the ßE202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the ß clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated ß clamp levels to impede growth and suggest either that multiple effects stemming from the dnaNE202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the ß clamp-Hda complex. The dnaNE202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaNE202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and ßE202K clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that ßE202K-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.


Asunto(s)
ADN Polimerasa III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Ribonucleósido Difosfato Reductasa/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas Bacterianas/genética , ADN Polimerasa III/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformación Proteica , Ribonucleósido Difosfato Reductasa/genética , Ribonucleótido Reductasas/genética
5.
Nucleic Acids Res ; 47(9): 4831-4842, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-30916336

RESUMEN

The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.


Asunto(s)
ADN Polimerasa III/genética , Replicación del ADN/genética , Proteínas de Escherichia coli/genética , Proteínas MutL/genética , Adenosina Trifosfatasas , Bacillus subtilis/química , Bacillus subtilis/genética , Sitios de Unión/genética , Reparación de la Incompatibilidad de ADN/genética , ADN Polimerasa III/química , Escherichia coli/genética , Modelos Moleculares , Proteínas MutL/química , Unión Proteica , Especificidad de la Especie
6.
Nucleic Acids Res ; 44(4): 1681-90, 2016 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-26657641

RESUMEN

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, ß. Single-molecule experiments reveal that the interactions of Pol II and Pol III with ß allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a ß-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.


Asunto(s)
ADN Polimerasa III/genética , ADN Polimerasa II/genética , ADN Polimerasa beta/genética , ADN/biosíntesis , ADN/genética , Daño del ADN/genética , ADN Polimerasa II/química , ADN Polimerasa III/química , ADN Polimerasa beta/química , Reparación del ADN/genética , Replicación del ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína
7.
PLoS Genet ; 11(9): e1005507, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26352807

RESUMEN

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the ß sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the ß clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.


Asunto(s)
Daño del ADN , ADN Polimerasa beta/metabolismo , Reparación del ADN , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Selección Genética , Dominio Catalítico , ADN Polimerasa beta/genética , Replicación del ADN , Escherichia coli/enzimología , Escherichia coli/metabolismo , Unión Proteica
8.
J Bacteriol ; 194(9): 2221-7, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22343299

RESUMEN

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of ß-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of ß-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.


Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Esporas Bacterianas/fisiología , Proteínas Bacterianas/genética , Operón Lac , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Viabilidad Microbiana , Subunidades de Proteína , Proteínas Recombinantes , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
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