Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail.

A variant of DNA polymerase β acts as a dominant negative mutant

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβΔ208–236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβΔ208–236 cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3ΔP cells were more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2′-hydrogen atoms with fluorine in the substrate 2′-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase–DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an ≈20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3′ side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.

Experimental surgery to create subgenomes of Bacillus subtilis 168

The 4,188-kb circular genome of Bacillus subtilis 168 was artificially dissected into two stable circular chromosomes in vivo, one being the 3,878-kb main genome and the other the 310-kb subgenome that was recovered as covalently closed circular DNA in CsCl-ethidium bromide ultracentrifugation. The minimal requirements to physically separate the 310-kb DNA segment out of the genome were two interrepeat homologous sequences and an origin of DNA replication between them. The subgenome originated from the 1,255–1,551-kb region of the B. subtilis genome was essential for the cell to survive because the subgenome was not lost from the cell. The finding that the B. subtilis genome has a potential to be divided and the resulting two replicons stably maintained may shed light on origins and formation mechanisms of giant plasmids or second chromosomes present in many bacteria. Similar excision or its reversal process, i.e., integration of large sized covalently closed circular DNA pieces into the main genome, implies significant roles of subgenomes in the exchange of genetic information and size variation of bacterial genomes in bacterial evolution.

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination–excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (≈40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli β-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

The Drosophila melanogaster Homologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

Uracil-DNA glycosylase–DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil–DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-Å resolution substrate analogue and 2.0-Å resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme–DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta

We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts.

CTXφ contains a hybrid genome derived from tandemly integrated elements

CTXφ is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5′ prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3′ element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXφ, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.

UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation

Plants are continuously subjected to UV-B radiation (UV-B; 280–320 nm) as a component of sunlight causing damage to the genome. For elimination of DNA damage, a set of repair mechanisms, mainly photoreactivation, excision, and recombination repair, has evolved. Whereas photoreactivation and excision repair have been intensely studied during the last few years, recombination repair, its regulation, and its interrelationship with photoreactivation in response to UV-B-induced DNA damage is still poorly understood. In this study, we analyzed somatic homologous recombination in a transgenic Arabidopsis line carrying a β-glucuronidase gene as a recombination marker and in offsprings of crosses of this line with a photolyase deficient uvr2–1 mutant. UV-B radiation stimulated recombination frequencies in a dose-dependent manner correlating linearly with cyclobutane pyrimidine dimer (CPD) levels. Genetic deficiency for CPD-specific photoreactivation resulted in a drastic increase of recombination events, indicating that homologous recombination might be directly involved in eliminating CPD damage. UV-B irradiation stimulated recombination mainly in the presence of photosynthetic active radiation (400–700 nm) irrespective of photolyase activities. Our results suggest that UV-B-induced recombination processes may depend on energy supply derived from photosynthesis.

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

Polarity of human replication protein A binding to DNA

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3′-end of the upstream oligonucleotide or at the 5′-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5′-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3′-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer–template systems containing 5′- or 3′-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.