Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis.

Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.

Analysis of the photoreceptors involved in the light-depending basidiocarp formation in Ustilago maydis.

We have described that formation of basidiocarps by Ustilago maydis requires illumination. In the current research, we have proceeded to analyze what kind of light receptors are involved in this phenomenon. Accordingly, we investigated whether the homologues of the White Collar (WC), and the phytochrome (PHY) genes played a role in this process. Mutants deficient in either one of the three U. maydis WC homologue genes (WCO1a, WCO1b, WCO2), or the phytochrome-encoding the PHY gene were obtained. Phenotypic analysis of the mutants showed that ∆wco1a mutants formed similar numbers of basidiocarps than wild-type strain, whereas ∆wco1b mutants were severely affected in basidiocarp formation when illuminated with white, blue or red light. ∆wco2 and ∆phy1 mutants did not form basidiocarps under any illumination condition. These data indicate that Wco1a is the main blue light receptor, and Wco1b may operate as a secondary blue light receptor; Phy1 is the red light receptor, and Wco2 the transcription factor that controls the photo stimulation of the genes involved in the formation of fruiting bodies. It is suggested that effectiveness of the light receptors depends on the whole structure of the complex, possibly, because their association is necessary to maintain their functional structure.

Compensatory role for Rad52 during recombinational repair in Ustilago maydis.

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.

Rec2 interplay with both Brh2 and Rad51 balances recombinational repair in Ustilago maydis.

Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.

Towards understanding the extreme radiation resistance of Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus exhibiting extreme resistance to UV and ionizing radiation. The molecular mechanisms underlying this resistance are as yet unknown. The recently determined genome sequence was examined for clues to the radiation resistance, focusing on proteins in homologous recombination, but there was little that was unusual about them. Furthermore, by comparison, its recombinational repair system seems to be only minimally related to the extended synthesis-dependent DNA strand-annealing system of Deinococcus radiodurans. Thus, consideration should be given to the possibility that incremental structural changes in repair proteins or their elevated expression are the basis for the extreme radiation resistance in U. maydis. Evolution of a system enabling the survival of U. maydis under such conditions could be a secondary consequence of adaptation to an environment of continual genotoxic stress encountered in its habitat.

Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis.

Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system.

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

White collar 1-induced photolyase expression contributes to UV-tolerance of Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus causing corn smut disease. It also is known for its extreme tolerance to UV- and ionizing radiation. It has not been elucidated whether light-sensing proteins, and in particular photolyases play a role in its UV-tolerance. Based on homology analysis, U. maydis has 10 genes encoding putative light-responsive proteins. Four amongst these belong to the cryptochrome/photolyase family (CPF) and one represents a white collar 1 ortholog (wco1). Deletion mutants in the predicted cyclobutane pyrimidine dimer CPD- and (6-4)-photolyase were impaired in photoreactivation. In line with this, in vitro studies with recombinant CPF proteins demonstrated binding of the catalytic FAD cofactor, its photoreduction to fully reduced FADH(-) and repair activity for cyclobutane pyrimidine dimers (CPDs) or (6-4)-photoproducts, respectively. We also investigated the role of Wco1. Strikingly, transcriptional profiling showed 61 genes differentially expressed upon blue light exposure of wild-type, but only eight genes in the Δwco1 mutant. These results demonstrate that Wco1 is a functional blue light photoreceptor in U. maydis regulating expression of several genes including both photolyases. Finally, we show that the Δwco1 mutant is less tolerant against UV-B due to its incapability to induce photolyase expression.

The excision of pyrimidine dimers from the DNA of mutant and wild-type strains of Ustilago.

UV-induced pyrimidine dimers were excised from the DNA of wild-type and four mutant strains of Ustilago maydis. Excision was partially dose dependent. The kinetics of excision differed in recombination deficient strains (rec 1 and rec 2) from those found in a recombination proficient radiation-sensitive strain (uvs 3). At fluences above 100 J-m-2 excision was saturated in uvs 3 but not in rec 1 or rec 2. Fluences above 300 J-m-2 started to saturate excision in wild-type. pol1-1, a temperature-sensitive DNA polymerase mutant, was excision proficient at both the permissive (22 degree) and restrictive (32 degree) temperatures. Wild-type cells were observed to excise CC before CT or TT in high dose experiments.

Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. I. Isolation and characterization of mutants.

The relationship between UV sensitivity and pyrimidine auxotrophy has been examined. Fourteen pyrimidine-requiring mutants have been classified on the basis of genetic complementation and utilization of biosynthetic intermediates and have been assigned to at least four loci. All the mutants studied were sensitive to UV, although the degree of sensitivity varied both between loci and amongst alleles at the same locus. A double mutant strain carrying pyrimidine mutants at two loci was only as sensitive to UV as the more sensitive of the singles. This suggests that both mutants are deficient in the same repair mechanism. Suppressor mutations which restored endogenous pyrimidine biosynthesis were isolated. These suppressors restored UV resistance in the presence of the original mutation. The results indicate that the UV sensitivity of these mutants is a direct result of pyrimidine auxotrophy and not to any secondary properties of the mutants.

Radiation-sensitive pyrimidine auxotrophs of Ustilago maydis. II. A study of repair mechanisms and UV recovery in pyr I.

Two mutants at the pyr I locus have been used to study the radiation sensitivity of pyrimidine auxotrophs of U. maydis. The mutant pry I-I has a reduced level of thymidine nucleotides, and this is a likely basis of the sensitivity. This strain is able to excise pyrimidine dimers from its DNA and is cross-sensitive to gamma-rays and nitrosoguanidine (NG) as well as to UV. A diploid heteroallelic at the pyr I locus was UV-sensitive but not deficient in UV-induced mitotic recombination. The results suggest that the UV sensitivity may be due to the failure of a repair DNA polymerase to fill post-excision single-strand gaps in the DNA. The mutant pyr I-I exhibits the property of UV recovery, and this is shown to be dependent on the presence of dimers in the DNA. A mechanism for UV recovery is proposed in which a repair system, possibly involving recombination, is induced by the UV irradiation.

Cloning the REC1 gene of Ustilago maydis.

The REC1 gene of Ustilago maydis plays a key role in homologous recombination and the repair of damaged DNA. In order to understand the nature and functions of the gene product, the gene has been cloned by functional complementation. A 3.8 kb cloned fragment complements the pleiotropic mitotic phenotype of different rec1 alleles. It does not complement the UV sensitivity of two other sensitive mutants. Disruption of the chromosomal copy of the 1.566 kb open reading frame within this fragment reproduces the rec1 pleiotropic phenotype. Furthermore, in diploids this disrupted reading frame is unable to complement previously characterised rec1 alleles.

Inducible DNA repair in Ustilago maydis.

Maximum survival of UV-irradiated U. maydis required a 2-3 h period of post-irradiation RNA and protein synthesis. Split dose experiments showed that this requirement correlated with the development of a radio-resistant cell state induced by UV doses above 200 Jm-2. Once induced, the radio-resistant state precluded the need for further RNA and protein synthesis for proficient repair of DNA damage caused by a second UV dose. Such radio-resistance was retained for up to 15 hours and it is concluded that this phenomenon represents the expression of an inducible DNA repair process, which is under transcriptional control.

DNA polymerase of Ustilago maydis: partial characterization of the enzyme and a pol 1 mutation.

The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive fro growth and DNA synthesis, and which at the restrictive temperature contains only 10-25% levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40 degrees C compared to that from the wild-type strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication.

Induced mutagenesis in Ustilago maydis. I. Isolation and characterization of a radiation-revertible allele of the structural gene for nitrate reductase.

A UV-revertible mutant of the nar1 structural gene for nitrate reductase was isolated in wild-type (nar+ nir+) Ustilago maydis. It proved to be vigorously revertible by gamma rays as well. Genetic analysis revealed that the strain carried a single, nonleaky, recessive allele (nar1-m) with an unusually high spontaneous reversion rate (approximately 3 X 10(-5)/div.). Reliable reversion frequencies were determined with a special agar medium that reduced the normally high level of residual growth observed on nitrate minimal agar. Radiation-induced reversion frequencies in the homozygous diploid were approximately twice those in the haploid. Following crosses to wild type, two revertants (one spontaneous and one UV-induced) were found to map at nar1. Although the molecular basis of nar1-m reversion is not known, available data suggest that some form of point mutation is involved.

Induced mutagenesis in Ustilago maydis. II. An in vivo biochemical assay.

UV gamma radiation-induced reversion to nar+ in a nar1-m nir1-1 strain of Ustilago maydis was found to occur under nongrowth conditions by performing the in vivo assay for functional nitrate reductase described by Resnick and Holliday (1971) who previously demonstrated that nonviable cells may still synthesize normal or near-normal levels of activity. Reversion frequencies of a signle gamma-irradiated culture were estimated in two cell populations by different methods: (A) among surviving clones after plating, and (B) among all cells (viable and nonviable) in suspension in the absence of postirradiation cell division. At gamma doses (300, 500 krad) corresponding to considerable cell killing (35%, 2% survival), reversion frequency by either method was the same. This supports the conclusion that mutation induction by gamma rays and its expression occur in nonviable cells with the same frequency as among survivors. If an error-prone repair system is assumed to be responsible for the observed gamma revertibility, then it is argued that this process is constituitive rather than inducible and that it is recombination-independent.