RESUMEN
In Escherichia coli, RecF-dependent post-replication repair (PRR) permits cells to tolerate the potentially lethal effects of blocking lesions at the replication fork. We have developed an in vivo experimental system to study the PRR mechanisms that allow blocked replication forks to be rescued by homologous sequences. We show that approximately 80% of the PRR events observed in SOS-uninduced cells are generated by RecA-mediated excision repair, a novel nucleotide excision repair- and RecA/RecF-dependent mechanism, while 20% are generated by RecF-dependent homologous recombination. Moreover, we show that in a wild-type background, PRR is approximately an order of magnitude more efficient in processing DNA containing a blocked leading strand, as compared with a blocked lagging strand. This strand bias is abolished in cells that are deficient in nucleotide excision repair. These results are discussed in the context of recent models describing the mechanisms of replication past damaged templates.
Asunto(s)
Reparación del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , PlásmidosRESUMEN
The Rad6/Rad18-dependent monoubiquitination of PCNA plays a crucial role in regulating replication past DNA damage in eukaryotic cells. We show here that in human cell-free extracts, efficient PCNA monoubiquitination requires both the synthesis of relatively long DNA tracts and polymerase idling or stalling at sites of DNA modification or DNA secondary structures. This dual dependency suggests a dynamic process in which, following initiation, the DNA synthesizing complex undergoes modifications that make it competent as a mediator for the activation of the Rad6/Rad18 pathway.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Sistema Libre de Células , Replicación del ADN , Células HeLa , HumanosRESUMEN
In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism - RecA-mediated excision repair - in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.
Asunto(s)
Reparación del ADN , ADN Bacteriano/biosíntesis , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Rec A Recombinasas , Medios de Cultivo , Daño del ADN , ADN Bacteriano/genética , Endodesoxirribonucleasas/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Plásmidos/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Transformación BacterianaRESUMEN
Lesions that transiently block DNA synthesis generate replication intermediates with recombinogenic potential. In order to investigate the mechanisms involved in lesion-induced recombination, we developed an homologous recombination assay involving the transfer of genetic information from a plasmid donor molecule to the Escherichia coli chromosome. The replication blocking lesion used in the present assay is formed by covalent binding of the carcinogen N-2-acetylaminofluorene to the C8 position of guanine residues (G-AAF adducts). The frequency of recombination events was monitored as a function of the number of lesions present on the donor plasmid. These DNA adducts are found to trigger high levels of homologous recombination events in a dose-dependent manner. Formation of recombinants is entirely RecA-dependent, the RecF and RecBCD sub-pathways accounting for about 2/3 and 1/3, respectively. Inactivation of recG stimulates recombinant formation about five-fold. In a recG background, the RecF pathway is stimulated about four-fold, while the contribution of the RecBCD pathway remains constant. In addition, in the recG strain, a recombination pathway that accounts for about 30% of the recombinants and requires genes that belong to both RecF and RecBCD pathways is revealed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Recombinación Genética , 2-Acetilaminofluoreno/farmacología , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Exodesoxirribonucleasa V/genética , Exodesoxirribonucleasa V/metabolismo , Regulación Bacteriana de la Expresión Génica , Guanina/metabolismo , Plásmidos/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Transducción de SeñalRESUMEN
The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/metabolismo , ADN/efectos de la radiación , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Genéticos , Proteínas Nucleares , Nucleotidiltransferasas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleotidiltransferasas/metabolismo , Secuencia de Bases , Línea Celular Transformada , Núcleo Celular/metabolismo , ADN/biosíntesis , ADN/genética , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Humanos , Técnicas In Vitro , Proteínas Nucleares , Nucleotidiltransferasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S , Técnicas del Sistema de Dos Híbridos , Rayos UltravioletaRESUMEN
Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.
Asunto(s)
Aminas/metabolismo , Aductos de ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Desoxiguanosina/análogos & derivadosRESUMEN
Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.
Asunto(s)
Unión Competitiva , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Replicación del ADN/genética , Escherichia coli/enzimología , Fragmentos de Péptidos/metabolismo , Cristalización , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa beta/genética , ADN Bacteriano/genética , Cinética , Ligandos , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , ADN Polimerasa III/fisiología , Replicación del ADN , Mutagénesis , Aductos de ADN/metabolismo , Reparación del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Respuesta SOS en GenéticaRESUMEN
Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/enzimología , Rec A Recombinasas/metabolismo , Recombinación Genética , ADN/genética , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Inhibición Psicológica , Mutación , Plásmidos , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/metabolismoRESUMEN
recX is a small open reading frame located downstream of recA that is conserved in many bacteria. In Escherichia coli, the recX gene (also named oraA) is a 501 bp open reading frame that encodes a predicted basic protein. Transcriptional analysis by Northern blots showed that in E. coli the recX gene is SOS-regulated. Primer extension data and transcriptional fusions indicate that recX transcription is down regulated with respect to recA by an intrinsic transcription terminator that is located between the recA and recX coding sequences. Despite the presence of this terminator, a recA-recX message resulting from transcriptional readthrough is detected at a level of 5-10% of the recA message. In addition, transcriptional/translational fusion experiments show that recX expression is further down regulated at the translational level reaching an estimated protein level about 500-fold lower than RecA. Strains in which the recX gene was disrupted were constructed by insertion of an antibiotic resistance cassette. The survival after UV irradiation, the spontaneous and UV-induced mutation rates were not significantly different in these recX strains compared to the corresponding wild type strain. Overexpression of RecA was shown to be lethal in a recX deletion strain in Pseudomonas aeruginosa [J. Bacteriol. 175 (1993) 2451], Mycobacterium tuberculosis [Mol. Microbiol. 30 (1998) 525] and Streptomyces lividans [J. Bacteriol. 182 (2000) 4005] suggesting that the recX gene may act as a regulator of recA. In contrast in E. coli, in a recX deletion strain, RecA overexpression is neither toxic nor is the expression of the recA(+) gene affected in a recX deletion strain at the basal level or after UV induction.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Rec A Recombinasas/genética , Northern Blotting , Cartilla de ADN , Mutación , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content. Despite the efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated. Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS). These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency. We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Mutagénesis/fisiología , Mutación , Nucleotidiltransferasas , Proteínas de Saccharomyces cerevisiae , Animales , Carcinógenos/toxicidad , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/fisiología , Escherichia coli/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Mutágenos/toxicidad , Moldes Genéticos , Xerodermia Pigmentosa/etiología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismoRESUMEN
Replication through (6-4)TT and G-AAF lesions was compared in Saccharomyces cerevisiae strains proficient and deficient for the RAD30-encoded DNA polymerase eta (Pol eta). In the RAD30 strain, the (6-4)TT lesion is replicated both inaccurately and accurately 60 and 40% of the time, respectively. Surprisingly, in a rad30 Delta strain, the level of mutagenic bypass is essentially suppressed, while error-free bypass remains unchanged. Therefore, Pol eta is responsible for mutagenic replication through the (6-4)TT photoproduct, while another polymerase mediates its error-free bypass. Deletion of the RAD30 gene also reduces the levels of both accurate and inaccurate bypass of AAF lesions within two different sequence contexts up to 8-fold. These data show that, in contrast to the accurate bypass by Pol eta of TT cyclobutane dimers, it is responsible for the mutagenic bypass of other lesions. In conclusion, this paper shows that, in yeast, translesion synthesis involves the combined action of several polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
In Escherichia coli nucleotide excision repair, the UvrB-DNA preincision complex plays a key role, linking adduct recognition to incision. We previously showed that the efficiency of the incision is inversely related to the stability of the preincision complex. We postulated that an isomerization reaction converts [UvrB-DNA], stable but incompetent for incision, into the [UvrB-DNA]' complex, unstable and competent for incision. Here, we identify two parameters, negative supercoiling and presence of a nick at the fifth phosphodiester bond 3' to the lesion, that accelerate the isomerization leading to an increasing incision efficiency. We also show that the [UvrB-DNA] complex is more resistant to a salt concentration increase than the [UvrB-DNA]' complex. Finally, we report that the [UvrB-DNA]' is recognized by UvrC. These data suggest that the isomerization reaction leads to an exposure of single-stranded DNA around the lesion. This newly exposed single-stranded DNA serves as a binding site and substrate for the UvrC endonuclease. We propose that the isomerization reaction is responsible for coupling UvrB and UvrC activities and that this reaction corresponds to the binding of ATP.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/metabolismo , Oligodesoxirribonucleótidos/metabolismo , 2-Acetilaminofluoreno/metabolismo , Adenosina Trifosfatasas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Isomerismo , Conformación de Ácido Nucleico , Plásmidos , Subunidades de ProteínaRESUMEN
We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3' end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol mu) was added. In contrast, purified human Pol mu alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol mu activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol mu (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.
Asunto(s)
2-Acetilaminofluoreno/metabolismo , Carcinógenos/metabolismo , Aductos de ADN/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Anticuerpos/farmacología , Secuencia de Bases , Extractos Celulares/análisis , Línea Celular Transformada , Células Cultivadas , ADN Polimerasa Dirigida por ADN/inmunología , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Inhibidores de la Síntesis del Ácido Nucleico , ARN Mensajero/biosíntesisRESUMEN
The biochemistry and genetics of translesion synthesis (TLS) and, as a consequence, of mutagenesis has recently received much attention in view of the discovery of novel DNA polymerases, most of which belong to the Y family. These distributive and low fidelity enzymes assist the progression of the high fidelity replication complex in the bypass of DNA lesions that normally hinder its progression. The present paper extends our previous observation that in Escherichia coli all three SOS-inducible DNA polymerases (Pol II, IV and V) are involved in TLS and mutagenesis. The genetic control of frameshift mutation pathways induced by N-2-acetylaminofluorene (AAF) adducts or by oxidative lesions induced by methylene blue and visible light is investigated. The data show various examples of mutation pathways with an absolute requirement for a specific combination of DNA polymerases and, in contrast, other examples where two DNA polymerases exhibit functional redundancy within the same pathway. We suggest that cells respond to the challenge of replicating DNA templates potentially containing a large diversity of DNA lesions by using a pool of accessory DNA polymerases with relaxed specificities that assist the high fidelity replicase.
Asunto(s)
ADN Polimerasa II/fisiología , ADN Polimerasa beta/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Escherichia coli/enzimología , Escherichia coli/genética , Mutagénesis/genética , 2-Acetilaminofluoreno/toxicidad , Carcinógenos/toxicidad , Aductos de ADN , Daño del ADN/efectos de los fármacos , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli , Mutación del Sistema de Lectura , Secuencia Rica en GC , Luz , Azul de Metileno/farmacología , Mutagénesis/efectos de la radiación , Oxidación-Reducción , Respuesta SOS en GenéticaRESUMEN
The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions. Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication. By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer. Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis. Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif. Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.
Asunto(s)
ADN Polimerasa II/fisiología , ADN Polimerasa beta/fisiología , Reparación del ADN , ADN Bacteriano/biosíntesis , ADN Polimerasa Dirigida por ADN/fisiología , Escherichia coli/enzimología , Mutagénesis , Mutación , Daño del ADN , Replicación del ADN/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli , Subunidades de ProteínaRESUMEN
The dinB-encoded DNA polymerase IV (Pol IV) belongs to the recently identified Y-family of DNA polymerases. Like other members of this family, Pol IV is involved in translesion synthesis and mutagenesis. Here, we show that the C-terminal five amino acids of Pol IV are essential in targeting it to the beta-clamp, the processivity factor of the replicative DNA polymerase (Pol III) of Escherichia coli. In vivo, the disruption of this interaction obliterates the function of Pol IV in both spontaneous and induced mutagenesis. These results point to the pivotal role of the processivity clamp during DNA polymerase trafficking in the vicinity of damaged-template DNA.