Genetic Analysis of the Hsm3 Protein Function in Yeast Saccharomyces cerevisiae NuB4 Complex

Abstract

In the nuclear compartment of yeast, NuB4 core complex consists of three proteins, Hat1, Hat2, and Hif1, and interacts with a number of other factors. In particular, it was shown that NuB4 complex physically interacts with Hsm3p. Early we demonstrated that the gene HSM3 participates in the control of replicative and reparative spontaneous mutagenesis, and that hsm3Δ mutants increase the frequency of mutations induced by different mutagens. It was previously believed that the HSM3 gene controlled only some minor repair processes in the cell, but later it was suggested that it had a chaperone function with its participation in proteasome assembly. In this work, we analyzed the properties of three hsm3Δ, hif1Δ, and hat1Δ mutants. The results obtained showed that the Hsm3 protein may be a functional subunit of NuB4 complex. It has been shown that hsm3- and hif1-dependent UV-induced mutagenesis is completely suppressed by inactivation of the Polη polymerase. We showed a significant role of Polη for hsm3-dependent mutagenesis at non-bipyrimidine sites (NBP sites). The efficiency of expression of RNR (RiboNucleotid Reducase) genes after UV irradiation in hsm3Δ and hif1Δ mutants was several times lower than in wild-type cells. Thus, we have presented evidence that significant increase in the dNTP levels suppress hsm3- and hif1-dependent mutagenesis and Polη is responsible for hsm3- and hif1-dependent mutagenesis.

The Role of Chromatin Assembly Factors in Induced Mutagenesis at Low Levels of DNA Damage

“…Previous studies have shown that the genes HAT1 , HIF1, and HSM3 , encoding subunits of the NuB4 complex, participated in the control of replicative and reparative spontaneous mutagenesis [ 36 , 55 , 58 ]. In these experiments, hat1Δ mutation epistatized to hsm3Δ and hif1Δ .…”

“…At high doses of UV, rad30Δ mutation completely suppressed hsm3 - and hif1 -specific mutagenesis [ 36 ]. We assumed that a similar interaction would also be observed in the asf1Δ rad30Δ double mutant.…”

“…Previously, we showed that mutations in HSM3 and HIF1 gene-encoding auxiliary subunits of the NuB4 increase the efficiency of UV-induced mutagenesis at high doses. However, hat1Δ and rad30Δ mutants showed a level of UV-induced mutagenesis at high doses similar to the wild-type strain [ 36 ].…”

“…LMG-3031 ( MATα ade2Δ-248 ura3-160,188 leu2-3,112 trp1 ) strain was transformed with those modules, and the transformants were selected on plates with selective media without uracil. The single rad52Δ (CAY-13), hsm6-1 (6B-SVK-312), pph3Δ (9-DVF-3031), hat1Δ (CAY-2), hsm3Δ (5-LMG-3031), hif1Δ (CAY-3), and rad30Δ (4-EAA-3031) strains were previously described [ 36 , 46 ]. The double asf1Δ rad52Δ (DVF-17), asf1Δ hat1Δ (DVF-18), asf1Δ hsm3Δ (DVF-19), asf1Δ hif1Δ (DVF-21), asf1Δ pph3Δ (DVF-22), and asf1Δ rad30Δ (TAE-156) mutants were obtained by replacement of ASF1 gene in rad52Δ , pph3Δ , rad53-HA-F (10-DVF-3031), hat1Δ , hsm3Δ , and hif1Δ strains, according to the same procedure.…”

“…Hif1p may play a role in depositing histones onto DNA, suggesting that Hat1p could be directly involved in the chromatin assembly process [ 30 ]. The NuB4 complex physically interacts with Hsm3p, which we previously identified as a functional subunit of the NuB4 complex [ 36 , 37 , 38 , 39 ]. Asf1p is linked to newly synthesized histones that bear the acetylation pattern characteristic of the Hat1p action [ 29 , 40 , 41 , 42 ].…”