A yeast-based genetic screening to identify human proteins that increase homologous recombination.
To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.
Collavoli
Anita
A
Laboratorio di Terapia Genica e Molecolare, Istituto di Fisiologia Clinica CNR, Area della Ricerca CNR Pisa, Italy.
Comelli
Laura
L
Rainaldi
Giuseppe
G
Galli
Alvaro
A
D004260:DNA Repair; D015723:Gene Library; D011905:Genes, ras; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000235:genetics; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
C104089:BASP1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D012097:Repressor Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D008741:Methyl Methanesulfonate; D046988:Proteasome Endopeptidase Complex