Academic journal article Genetics

The RAD6/BRE1 Histone Modification Pathway in Saccharomyces Confers Radiation Resistance through a RAD51-Dependent Process That Is Independent of RAD18

Academic journal article Genetics

The RAD6/BRE1 Histone Modification Pathway in Saccharomyces Confers Radiation Resistance through a RAD51-Dependent Process That Is Independent of RAD18

Article excerpt

ABSTRACT

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Δ, lge1Δ, and rtf1Δ, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Δ mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Δ mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Δ, bre1Δ, and lge1Δ mutants show epistasis for IR sensitivity. The paf1Δ mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Δ, rad51null, rad50Δ, and rad9Δ mutations are epistatic to bre1Δ and dot1Δ, but rad18Δ and rad5Δ show additivity with bre1Δ, dot1Δ, and each other. The bre1Δ rad18Δ double mutant resembles rad6Δ in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Δ. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G^sub 1^ checkpoint role for the RAD6/BRE1/DOT1 pathway.

RECENT research in eukaryotes has demonstrated a much greater role than was initially perceived for histone modifications in basic cellular processes, including transcription, gene silencing, control of carcinogenesis, and responses to DNA damage. As part of this, we reported that Saccharomyces strains deleted for any of several genes involved in histone modifications are substantially more sensitive than wild type to the lethal effects of ionizing radiation (IR) (GAME et al. 2005). The mutants included strains deleted for the DOT1 gene, which encodes the methylase that acts on the lysine 79 residue (K79) of the histone H3 protein (FENG et al. 2002; VAN LEEUWEN et al. 2002), as well as histone H3 mutants in which wild-type Dot1p cannot act because its target lysine is replaced with another amino acid. These findings complemented information from other laboratories that implicates histone H3 lysine 79 methylation in controlling the DNA damage checkpoint induced by ultraviolet radiation and other agents in yeast (GIANNATTASIO et al. 2005; WYSOCKI et al. 2005) and in damage recognition by the checkpoint protein 53BP1 in mammalian cells (HUYEN et al. 2004).

Substantial information is available indicating that the DOT1-mediated methylation of H3 K79 is dependent on the prior modification of histone H2B involving ubiquitination of lysine 123 in Saccharomyces (BRIGGS et al. 2002; NG et al. 2002a) or lysine 120 in mammals (KIM et al. 2005). Recently, it was shown that H3 K79 trimethylation and some di-methylation is dependent on H2B K123 ubiquitination, whereas mono-methylation of K79 still occurs fully even in mutants that fail to modify H2B K123 (SHAHBAZIAN et al. 2005). The Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase together ubiquitinate H2B K123 (ROBZYK et al. 2000; HWANG et al. 2003; WOOD et al. 2003a). In addition, the LGE1 gene product has been found to complex with Bre1 protein and is required for its function (HWANG et al. 2003), and mutants involving some members of the RNA polymerase II-associated PAF1 complex, specifically deletions of the RTF1 and PAF1 genes, have also been reported to abolish H2B K123 ubiquitination (NG et al. 2003a; WOOD et al. 2003b). Most recently, the Bur1/ Bur2 cyclin-dependent protein kinase has also been implicated in H2B K123 ubiquitination through its role in activating the Rad6 protein by phosphorylation (WOOD et al. 2005).

Given this information, and to better understand the role of the RAD6 gene in different DNA repair pathways, we chose to study the X-ray sensitivity of additional Saccharomyces histone modification mutants, including those with reported defects in H2B K123 ubiquitination and H3 K79 methylation and those involved in methylation elsewhere on histone H3. …

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