SetRICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response.

The integrating conjugative element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA polymerase V ortholog, polVICE391, encoded by the ICE391 rumAB operon. polV and its orthologs have previously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in vivo. As a result, multiple levels of regulation are imposed on the polymerases so as to avoid aberrant mutagenesis. We report here, that the mutagenesis-promoting activity of polVICE391 is additionally regulated by a transcriptional repressor encoded by SetRICE391, since Escherichia coli expressing SetRICE391 demonstrated reduced levels of polVICE391-mediated spontaneous mutagenesis relative to cells lacking SetRICE391. SetRICE391 regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetRICE391 revealed that under alkaline conditions, as well as in the presence of activated RecA, SetRICE391 undergoes a self-mediated cleavage reaction that inactivates repressor functions. Conversely, a non-cleavable SetRICE391 mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polVICE391-dependent mutagenesis. Electrophoretic mobility shift assays revealed that SetRICE391 acts as a transcriptional repressor by binding to a site overlapping the -35 region of the rumAB operon promoter. Our study therefore provides evidence indicating that SetRICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response.

DNA polymerase ι: The long and the short of it!

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.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation.

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli.

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD'2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the "steric gate" residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical "nick translation". In vitro, pol I displaces 1-3 nucleotides of the RNA/DNA hybrid and through its 5'→3' (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the "nick-translation" activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th) phosphodiester bond 5' and 4(th)-5(th) phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER.

A strategy for the expression of recombinant proteins traditionally hard to purify.

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.