BMC Genetics BioMed Central Research article Open Access Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast Elena I Stepchenkova1, Stanislav G Kozmin1,2, Vladimir V Alenin1 and Youri I Pavlov*1,3 Address: 1Department of Genetics, Sankt-Petersburg State University, Sankt-Petersburg, 199034, Russia, 2Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, RTP, NC 27709, USA and 3Eppley Institute for Research in Cancer and Allied Diseases, the Department of Biochemistry and Molecular Biology, and the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA Email: Elena I Stepchenkova - [email protected]; Stanislav G Kozmin - [email protected]; Vladimir V Alenin - [email protected]; Youri I Pavlov* - [email protected] * Corresponding author Published: 02 June 2005 Received: 26 January 2005 Accepted: 02 June 2005 BMC Genetics 2005, 6:31 doi:10.1186/1471-2156-6-31 This article is available from: http://www.biomedcentral.com/1471-2156/6/31 © 2005 Stepchenkov et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino- 6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA. Background [1-4]. Such modified bases may have ambiguous base- The accurate replication and repair of genetic material, pairing properties that will result in a high mutagenic which is a prerequisite for normal functioning of the activity after their incorporation into DNA during replica- eukaryotic genome and the prevention of cancer, relies on tion. A classic example of the detoxification mechanism is coordinated and faithful DNA synthesis. One important the elimination of dUTP and 8-oxo-dGTP from the dNTP mechanism that ensures a high fidelity of DNA replication pool by the E. coli dUTPase and MutT proteins, respec- is a "cleansing" of the DNA precursor pool from deoxyri- tively [1,5]. bonucleoside triphosphates containing a modified base Page 1 of 11 (page number not for citation purposes) BMC Genetics 2005, 6:31 http://www.biomedcentral.com/1471-2156/6/31 H H tivity to HAP in yeast due to mutations in the HAM1 gene N O [18]. When we cloned and sequenced the HAM1 gene, we N N N N found that it has homologs in many organisms, from bac- O teria to humans [13], and proposed that the gene might N N N N H N N N code for new triphosphatase [16]. Then, the crystal struc- H H H ture of the Ham1p homologue from a thermostable bac- Adenine N N Guanine terium (protein Mj0226) was determined [19]. It was H found that the Ham1p ternary structure has common fea- Hypoxanthine tures with MutT. Homologs of the yeast Ham1p from H OH H OH other organisms possessed triphosphatase activity on N N dITP, ITP, XTP, and dHAPTP substrates (Kozmin and Pav- N N N N lov, unpublished; Burgis and Cunnigham, personal com- munication; and [19-21]). N N H N N N H H H There are additional, less thoroughly studied, factors modulating purine base analogues mutagenesis in yeast 6-Hydroxylaminopurine 2-Amino-6-Hydroxylaminopurine (see [16] for review). For example, aah1 mutants are sen- sitive to HAP, suggesting that adenine deaminase Aah1p ChemicalbasesFigure 1 structures of HAP and AHA and natural purine may deaminate HAP base to hypoxanthine [16]. Chemical structures of HAP and AHA and natural purine bases. In the present study, we carried out a genome-wide search for HAP and AHA sensitive mutants. The release of several complete sets of deletion mutants by the Yeast Deletion Project provides a powerful approach for different types of genome screens in yeast [22]. Haploid and diploid strains Purine analogs 6-hydroxyaminopurine (HAP) and 2- have already been used to detect new genes controlling amino-HAP (AHA) are powerful mutagens in bacteria, sensitivity to different agents such as UV, ionising radia- yeast, and higher eukaryotes [6,7]. It has been suggested tion, iron, and methyl methane sulfonate (MMS) [23-27], that HAP-deoxyriboside-triphosphate (dHAPTP) is a pos- as well as spontaneous mutability [28]. This approach, sible endogenous contaminant of nucleotide pools under when combined with other genomics approaches, helps peroxyl radical stress [8]. HAP and AHA closely resemble to establish the biological functions of uncharacterized the natural purines, hypoxanthine and xanthine (Fig. 1), ORFs in yeast, many of which have human orthologs. This and therefore, could be exploited to investigate the mech- approach also allows us to decipher the network anism preventing mutations that are caused by non- responses to endogenous and environmental stress [29]. canonical purine nucleotides [9-11]. The present study is the first systematic, genome-wide search for the mutations conferring sensitivity to muta- It was proposed that purine salvage enzymes convert base genic purine base analogs. analogs to the corresponding deoxyribonucleoside tri- phosphates, which are misincorporated or misreplicated Results during DNA synthesis, resulting in induction of muta- Development of the screening method tions [12,13]. HAP-induced mutagenesis in yeast is ele- To develop a useful method for searching the yeast vated in strains with defects in proofreading activity of mutants sensitive to base analogs, we calibrated the exper- replicative DNA polymerases [14,15] and does not imental conditions using the wild-type strain, BY4742, depend on excision, mutagenic recombination, and mis- and two previously described HAP-sensitive mutants, match repair systems [14-16]. We have described several ham1 and aah1 (see [6,16]), created on BY4742 back- systems protecting cells from the mutagenic and ground. As shown in Fig. 2 and described in Materials and inhibitory effects of HAP (see review [16]). One is the Methods, yeast were grown in a 96-well microtiter plate novel molybdenum cofactor-dependent system in E. coli and then transferred, using a multiprong replicator device, [17]. It has yet to be determined if a similar system exists to YPD plates containing base analogs. An induction of in higher eukaryotes. Another, versatile HAP-detoxifica- the Canr mutants was monitored by replica-plating to the tion pathway relies upon the action of triphosphatase, minimal-medium plates containing L-canavanine. Ham1p, which hydrolyze HAP-containing ribo- and deoxyribo-nucleotides to nucleoside monophosphates, The results are presented in Fig. 3. The left panel of Fig. 3 and which prevent incorporation of base analog into shows the induction of canavanine-resistant mutants by DNA and RNA. We initially described the elevated sensi- HAP and AHA; and the right panel represents the survival Page 2 of 11 (page number not for citation purposes) BMC Genetics 2005, 6:31 http://www.biomedcentral.com/1471-2156/6/31 Master plate Growth of the yeast strains on YPD medium YPD YPD YPD with HAP 1 µg/ml HAP 10 µg/ml HAP 100 µg/ml base analogs Dilution or without any mutagens YPD YPD YPD AHA 10 µg/ml AHA 100 µg/ml Replica plating on selective SD medium Replica plating YPD of dilutions HAP 1 µg/ml YPD YPD on YPD medium HAP 10 µg/ml HAP 100 µg/ml with HAP, AHA or YPD without any AHA 100 µg/ml mutagens SD, L-canavanine 40 mg/l YPD YPD AHA 10 µg/ml Test for the induction of Canr mutants Survival test by the base analogs HAP and AHAP SchemeFigure 2of the protocol for screening the yeast deletions library for base analog sensitivity and induced mutagenesis Scheme of the protocol for screening the yeast deletions library for base analog sensitivity and induced mutagenesis. of the tested strains on YPD plates in the presence of base ant clones at 10 and 100 µg/ml of HAP, in comparison analogs. In the wild-type strain, as in the ham1 and aah1 with 1 µg/ml of HAP observed in the ham1 mutant (Fig. mutants, 1–3 spontaneous canavanine-resistant colonies 3B, left panel, another manifestation of hypersensitivity per spot arise in the absence of mutagen (Fig. 3A and 3B, phenotype, HS), is also due to a dramatic decrease of sur- left panel). In our experimental conditions, 1 µg/ml of vival. When the aah1 mutant was tested (Fig. 3B, second HAP did not induce Canr mutants in the wild-type strain. row, for HAP and Fig. 3F, first row, for AHA), the drop of A moderate induction of Canr clones (fewer than ten per viability was less severe than that for the ham1 mutant spot) was observed at 10 µg/ml of HAP and a very strong (phenotype of elevated sensitivity, S).