Both the charged linker region and ATPase domain of Hsp90 are essential for Rad51-dependent DNA repair.
The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time.
Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Tanvi
T
Suhane
Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Shyamasree
S
Laskar
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Siddheshwari
S
Advani
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Nabamita
N
Roy
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Shalu
S
Varunan
Tata Memorial Centre, ACTREC, Mumbai, India.
Dibyendu
D
Bhattacharyya
Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India sbtsl@uohyd.ernet.in mkbsl@uohyd.ernet.in.
Sunanda
S
Bhattacharyya
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India sbtsl@uohyd.ernet.in mkbsl@uohyd.ernet.in.
Mrinal Kanti
MK
Bhattacharyya
D020816:Amino Acid Motifs; D020134:Catalytic Domain; D004260:DNA Repair; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D051135:Rad51 Recombinase / Q000378:metabolism*; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C074510:HSP82 protein, S cerevisiae; D018841:HSP90 Heat-Shock Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase