Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an 7 kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB_(R394) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a ΔumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.     

poliota-dependent lesion bypass in vitro

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polκ)-like; Rev1-like, Rad30A (polη)-like and Rad30B (polι)-like polymerases. The polIV/polκ-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and polη-like polymerases are found exclusively in eukaryotes, and to date, polι-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the polι-like polymerases, where the enzymatic properties of Drosophila melanogaster polι are more similar to that of Saccharomyces cerevisiae and human polη than to the related human polι. For example, like polη, Drosophila polι can bypass a cis-syn thymine–thymine dimer both accurately and efficiently, while human polι bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human polι can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3′T of a 6-4-thymine–thymine pyrimidine–pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although polι most likely arose from a genetic duplication of polη millions of years ago as eukaryotes evolved, it would appear that polι from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have “tailored” its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian polι plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for polι and the identification of mice deficient in polι.     

Elevated expression of DNA polymerase II increases spontaneous mutagenesis in Escherichia coli

Escherichia coli DNA polymerase II (Pol-II), encoded by the SOS-regulated polB gene, belongs to the highly conserved group B (-like) family of “high-fidelity” DNA polymerases. Elevated expression of polB gene was recently shown to result in a significant elevation of translesion DNA synthesis at 3, N⁴-ethenocytosine lesion with concomitant increase in mutagenesis. Here, I show that elevated expression of Pol-II leads to an approximately 100-fold increase in spontaneous mutagenesis in a manner that is independent of SOS, umuDC, dinB, recA, uvrA and mutS functions. Cells grow slowly and filament with elevated expression of Pol-II. Introduction of carboxy terminus (“β interaction domain”) mutations in polB eliminates elevated spontaneous mutagenesis, as well as defects in cell growth and morphology, suggesting that these abilities require the interaction of Pol-II with the β processivity subunit of DNA polymerase III. Introduction of a mutation in the proofreading exo motif of polB elevates mutagenesis by a further 180-fold, suggesting that Pol-II can effectively compete with DNA polymerase III for DNA synthesis. Thus, Pol-II can contribute to spontaneous mutagenesis when its expression is elevated.     

Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes

We focused our attention on the isolation and regulation of genes encoding for pectin methylesterase (PME) isoenzymes in Arabidopsis thaliana (At). The present data reports the identification of two PME-like genes, named AtPME1 and AtPME2, that are closely linked in the At genome. These genes possess different structural organisations. While all higher plant PME characterised so far possess an intron at a similar location relative to their coding sequence, AtPME2 shows an additional intron whose presence within other higher plant PME-like genes has not been previously reported. Sequence comparison of the N-terminal region suggests that the secretory pathways of the putative AtPME1 and AtPME2 isoforms are different, and that this region may contribute to specify a biological function to each isoform. Moreover, phylogenetic analysis reflects the possible existence of functional groups of PME isoforms in higher plant species that seem to have been conserved during evolution.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

Sequence analysis of three family B DNA polymerases from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis.

Three family B DNA polymerase genes, designated B1, B2, and B3, were cloned from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis, and sequenced. Deduced amino acid sequences of B1 and B3 DNA polymerases have all exonuclease and polymerase motifs which include critical residues for catalytic activities. Furthermore, a YxGG/A motif, which is located between 3'-5' exonuclease and polymerization domains of family B DNA polymerases, was also found in each of the B1 and B3 sequences. These findings suggested that S. ohwakuensis B1 and B3 DNA polymerases have both exonuclease and polymerase activities. However, amino acid sequence of the B2 DNA polymerase of this organism contains several amino acid substitutions in Pol-motifs, and also lacks Exo-motif I and Exo-motif II. These substitutions and lack of certain motifs raise questions about polymerase and exonuclease activities of the corresponding gene product. The B3 sequence of S. ohwakuensis is more closely related to Pyrodictium, Aeropyrum, and Archaeoglobus DNA polymerase B3 sequences than to the Sulfolobus B3 sequences. Phylogenetic analysis showed that crenarchaeal B1 DNA polymerases are closely related to each other, and suggested that crenarchaeal B3, euryarchaeal family B, and eukaryal epsilon DNA polymerases may be orthologs.     

Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Nucleotide sequence of the transposable element IS15

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-Δ, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-Δ possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental 1S15-Δ.     

The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control

Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase ζ, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed.     

Current understanding of UV-induced base pair substitution mutation in E. coli with particular reference to the DNA polymerase III complex

UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photorevesal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective ε proofreading subunit) had any effect on he misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of ε proofreading activity by recA protein are possible.

In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3′–5′ proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.     

The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis.

In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

Vertebrate immunity: mutator proteins and their evolution

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.     

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.     

武夷山自然保护区土壤可培养芽胞杆菌 的物种多样性及分布

为了解武夷山自然保护区土壤中可培养芽胞杆菌的分布状况, 2012年6月从该保护区的黄岗山顶部、中部、底部和桐木关、挂墩、大竹岚等6个地点采集土样75份。用80℃水浴加热、稀释平板法进行芽胞杆菌的分离, 并根据16S rRNA基因序列分析对菌株进行初步鉴定。从土样中分离出芽胞杆菌418株, 鉴定为8个属42个种, 其中Bacillus属的种数最多, 有20种, Paenibacillus属和Lysinibacillus属分别有8种和7种。不同地点分离到的芽胞杆菌在种类、数量上存在差异: 从大竹岚土壤中分离到的芽胞杆菌种类最多, 从黄岗山中部和底部分离到的种类数则较少; 挂墩、大竹岚土壤中芽胞杆菌的数量较大, 达3.6×10⁶ cfu/g以上, 而黄岗山顶部和中部土壤中的数量则少于4.9×10⁵ cfu/g。Bacillus cereus、B. mycoides、B. thuringiensis和Lysinibacillus xylanilyticus等4个种在6个地点的土样中均有分离到, 其中B. thuringiensis、L. xylanilyticus是该保护区土壤中的优势种。桐木关土壤中芽胞杆菌的种类多样性和均匀度指数都比其他5个地点的高, 而挂墩土壤中芽胞杆菌的Shannon-Wiener多样性、均匀度和优势度指数都最低。B. mycoides和B. thuringiensis的数量与海拔显著相关, 相关系数分别为0.852和-0.834, B. cereus、B. mycoides、B. thuringiensis的分离频度与海拔的相关性极显著, 相关系数分别为0.960、0.952和-0.931。研究结果表明, 武夷山自然保护区土壤中可培养芽胞杆菌的种类丰富、数量较大, 具有较高的多样性。     

The Cryphonectria parasitica plasmid pUG1 contains a large ORF with motifs characteristic of family B DNA polymerases.

The isolation and characterization of the circular mitochondrial plasmid pUG1 from the ascomycete Cryphonectria parasitica is described. The entire sequence (4182 bp) was obtained and high similarities to DNA-dependent DNA polymerases were revealed. Strikingly common features with the DNA polymerases encoded by the Neurospora intermedia plasmids Fiji and LaBelle, such as matches to the conserved motifs A and B and the presence of TTD instead of DTD in motif C, were found, suggesting the existence of a distinct group of members of the B DNA family polymerases. These strong similarities between the plasmids might suggest a common origin of the C.parasitica and the Neurospora plasmids.