RESUMEN
The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform.
Asunto(s)
Codón Iniciador , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Secuencia de Aminoácidos , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Isoenzimas , Ubiquitinación , ADN Polimerasa iotaRESUMEN
Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I-V. For example, in a recA730 lexA(Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Mutagénesis , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutación MissenseRESUMEN
Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to
Asunto(s)
ADN Polimerasa Dirigida por ADN/deficiencia , Mutación/efectos de la radiación , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Secuencia de Aminoácidos , Línea Celular , ADN Polimerasa Dirigida por ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Fibroblastos , Humanos , Datos de Secuencia Molecular , Transfección , Rayos Ultravioleta , Xerodermia Pigmentosa/patología , ADN Polimerasa iotaRESUMEN
Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV(R391), a polV-like Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon R391. When expressed in a DeltaumuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively. Analysis of the spectrum of polV(R391)-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G-->A:T and A:T-->T:A transversions at certain mutagenic hot spots. Biochemical characterization of polV(R391) highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polV(R391) can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3'T of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA'B, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.
Asunto(s)
Elementos Transponibles de ADN , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutagénesis , Respuesta SOS en GenéticaRESUMEN
Y-family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error-prone than high-fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer-termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of poliota (P692R) and poleta (H654A) were isolated that are defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.