Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability in Saccharomyces cerevisiae

Abstract

A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue.

Keywords: cryptic transcription; histone assembly; nucleosome; nucleotide excision repair.

Figure 1

Growth phenotypes of single sprocket arginine histone mutants. (A) Location of the sprocket arginine residues is highlighted in pink in the yeast nucleosome structure (1id3) (White et al. 2001). (B and C) Yeast strains containing a single histone arginine mutation were spotted at decreasing serial dilutions on SC-LEU and SC-LEU+5-FOA plates. The SC-LEU+5-FOA plates select against a URA3 plasmid containing wild-type histones, revealing the growth phenotype of the histone mutant. SHL, superhelical location.

Figure 2

Effects of histone sprocket arginine mutants on expression of the HO-lacZ reporter and cryptic transcription of the FLO8-HIS3 reporter. (A) LacZ activity was measured for extracts from each histone mutant strain in a snf5∆ genetic background. Mean and standard deviation of LacZ activity from at least three independent experiments is depicted for each strain. The SHLs of nucleosomal DNA bound by each arginine residue are indicated. P-values were corrected for multiple hypothesis testing. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. ªThe H4 R45A mutant strain also contains the wild-type (wt) histone H4 gene. (B) Same as in A, except the strains are wild type for SNF5. (C) Yeast strains containing the FLO8-HIS3 reporter (excepting the wild-type FLO8 control) were spotted at decreasing serial dilutions on SC-LEU and SC-LEU-HIS plates. Growth on SC-LEU-HIS plates indicates activation of the FLO8 cryptic transcript containing the HIS3 reporter. ^bThe H4 R45H mutant strain also contains the wild type (wt) histone H4 gene.

Figure 3

H3 R49 and H2A R78 histone residues are required for survival and efficient NER of UV-induced DNA lesions. (A) UV survival assays of histone mutant strains. (B) Representative alkaline gels from genomic NER assays of histone mutants. Genomic DNA was isolated from yeast cells before UV exposure or at the indicated time points following UV radiation (100 J/m²). Isolated genomic DNA was treated with or without T4 endonuclease, which makes single-stranded DNA (ssDNA) nicks at sites of UV-induced CPDs. (C) Quantification of initial frequency of CPDs per kilobase of genomic DNA. (D) Quantification of the percentage of repair of CPDs from alkaline gels. Mean and standard deviation of the percentage of repair of CPDs from three independent experiments is depicted.

Figure 4

Histone sprocket arginine mutants are sensitive to MMS, but do not affect BER efficiency of MMS-induced DNA lesions. (A) Sprocket arginine mutant strains were spotted at decreasing serial dilutions on plates containing the indicated doses of MMS. (B) Representative alkaline gels from genomic BER assays of histone mutants and a Δmag1 control strain. DNA was isolated from yeast cells before MMS exposure or at the indicated time points following MMS treatment (0.2% for 10 min). Isolated genomic DNA was treated with (or without) AAG/APE1 to induce ssDNA nicks at sites of 3-methyladenine and 7-methylguanine lesions. (C) Quantification of initial frequency 3-methyladenine and 7-methylguanine lesions per kilobase of genomic DNA. (D) Quantification of the percentage of repair of MMS-induced DNA damage from alkaline gels. Mean and standard deviation of the percentage of repair from three independent experiments is depicted.

Figure 5

The histone H2A R78A-H2B R36A double mutant causes a rapid cessation of cell growth and a defect in histone occupancy. (A) Yeast strains containing the indicated double mutants were spotted as described in Figure 1B. (B) Growth rate of histone mutants upon repression of wild-type histone H2A and H2B genes by shifting to glucose media (“0” hour). The H2A ∆L1 strain has a deletion in the essential L1 loop (residues 39–42). (C) Histone occupancy of the FLAG-tagged wild-type or mutant histone H2B in the indicated genetic background (see text for more details) was measured by ChIP at select genomic regions. *Strain contained wild-type H2A and H2A R78A mutant. (D) Same as in C using a FLAG-tagged wild-type H2B or mutant H2B L103R.

Figure 6

Translational positions of sprocket arginine residues, their associated DNA-binding motifs, and their mutant phenotypes, as identified by this study. The yeast nucleosome structure (1id3) (White et al. 2001) was displayed using PyMOL.