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. 2015 Sep:33:70-7.
doi: 10.1016/j.dnarep.2015.05.011. Epub 2015 Jun 19.

LAMMER kinase contributes to genome stability in Ustilago maydis

Carmen de Sena-Tomás  1 , Jeanette H Sutherland  1 , Mira Milisavljevic  2 , Dragana B Nikolic  2 , José Pérez-Martín  3 , Milorad Kojic  4 , William K Holloman  5
Affiliations

LAMMER kinase contributes to genome stability in Ustilago maydis

Carmen de Sena-Tomás et al. DNA Repair (Amst). 2015 Sep.

Abstract

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.

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Figures

Fig. 1
Fig. 1
Phenotype of Muv3. The Muv3 mutant was tested for sensitivity to UV, MMS, and HU and compared with the brh2Δ and rad51Δ mutants. Spot tests were performed at least three times with independent isolates. Elongated cellular morphology evident in Muv3, brh2Δ, and rad51Δ mutants is illustrated in the micrographs by the arrows and was tallied as shown after counting 400-500 cells of each strain under a microscope using a hemocytometer.
Fig. 2
Fig. 2
Cloning by complementation. Two classes of clones were obtained after introducing a genomic library into the Muv3 mutant. Representative clone 1 and clone 2 were compared for ability to rescue the sensitivity to UV, MMS, and HU. The two different DNA fragments isolated are illustrated with genes present according to the annotated MIPS database. Subclones were prepared from clone 1 with the restriction enzyme fragments indicated. Spot tests were performed on three independent isolates.
Fig. 3
Fig. 3
Lkh1 in U. maydis. A. Dendrogram of LAMMER kinases. The tree was constructed using the ClustalW method (http://embnet.vital-it.ch/software/ClustalW.html). Bar scale = 0.05 substitutions per amino acid. The proteins utilized were Saccharomyces cerevisiae (Sc) Kns1 (CAA97468); Schizosaccharomyces pombe (Sp) Lkh1 (CAD29835); Homo sapiens (Hs) LKH1/CLK3 (NP_001123500); Drosophila melanogaster (Dm) Doa (P49762); Arabidopsis thaliana (At) Ame3/Afc1 (P51568); U. maydis (Um) Lkh1 (KP966103). B. Lkh1 illustrated schematically with Kinase domain as gray bar and motifs for ATP binding, S/T kinase signature, and LAMMER signature motif in dark gray. The arrow indicates the site of mutation to a stop codon. C. Comparison of survival of lkh1 alleles. Spot tests were performed at least three times.
Fig. 4
Fig. 4
Suppression of genotoxin sensitivity by expression of core HR components. Plasmids expressing Brh2, Rad51, or Dss1 (e.g., indicated as + Brh2, etc.) were introduced into wild type, lkh1Δ, and lkh1Q488k strains. Survival was assessed after treatment as shown. Spot tests were performed at least three times with independent isolates.
Fig. 5
Fig. 5
Epistasis and cell cycle effects. A. lkh1Q488k was examined for epistasis with rad51Δ. B. Comparison of lkh1Q488k phenotype with DNA damage checkpoint and HR mutants. C. lkh1 alleles were examined for epistasis with chk1Δ. D. FACS scans were performed on exponentially growing cultures that were treated with 50 mM HU. Samples were removed for analysis at 2 hr intervals after addition of HU. Survival spot tests were performed at least three times with independent isolates. FACS analyses were performed with at least three different isolates for each strain and on two separate occasions.
Fig. 6
Fig. 6
lkh1 meiotic phenotype. A. Heteroallelic recombination at nar1 was measured in meiotic progeny from germinated teliospores. In the case of wild type Nar+ recombinants were determined after105 cells were plated on each of two plates. In the case of the lkh1 alleles Nar+ was determined after 106 cells were plated on each of 4 plates. Standard deviations are shown. B. Meiotic progeny were spread on charcoal medium to measure Fuz phenotype. Representative plates are shown for wild type and lkh1 alleles. 500-1000 colonies were tallied for each strain. Percentage of Fuz+ is shown underneath. C. FACS scan analysis of representative meiotic progeny from the crosses shown. For the lkh1Δ and lkh1Q488k alleles Fuz+ and Fuz− colonies were chosen at random. FACS analysis was performed on two independent matings with 10 different meiotic product isolates for each genotype.
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