Recruitment of HRDC domain of WRN and BLM to the sites of DNA damage induced by mitomycin C and methyl methanesulfonate.
The HRDC (helicase and RNase D C-terminal) domain at the C-terminal of WRNp (Werner protein) (1150-1229 amino acids) and BLMp (Bloom protein) (1212-1292 amino acids) recognize laser microirradiation-induced DNA dsbs (double-strand breaks). However, their role in the recognition of DNA damage other than dsbs has not been reported. In this work, we show that HRDC domain of both the proteins can be recruited to the DNA damage induced by MMS (methyl methanesulfonate) and MMC (methyl mitomycin C). GFP (green fluorescent protein)-tagged HRDC domain produces distinct foci-like respective wild-types after DNA damage induced by the said agents and co-localize with γ-H2AX. However, in time course experiment, we observed that the foci of HRDC domain exist after 24 h of removal of the damaging agents, while the foci of full-length protein disappear completely. This indicates that the repair events are not completed by the presence of protein corresponding to only the HRDC domain. Consequently, cells overexpressing the HRDC domain fail to survive after DNA damage, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Moreover, 24 h after removal of damaging agents, the extent of DNA damage is greater in cells overexpressing HRDC domain compared with corresponding wild-types, as observed by comet assay. Thus, our observations suggest that HRDC domain of both WRN and BLM can also recognize different types of DNA damages, but for the successful repair they fail to respond to subsequent repair events.
Department of Life Science and Biotechnology, Jadavpur University, Kolkata, West Bengal, India.
Saheli
S
Samanta
Parimal
P
Karmakar
D000970:Antineoplastic Agents / Q000009:adverse effects*; D004249:DNA Damage / Q000187:drug effects*; D004265:DNA Helicases / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008741:Methyl Methanesulfonate / Q000009:adverse effects*; D016685:Mitomycin / Q000009:adverse effects*; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry; D014162:Transfection; D000071657:Werner Syndrome Helicase
D000970:Antineoplastic Agents; D016685:Mitomycin; D008741:Methyl Methanesulfonate; D005090:Exodeoxyribonucleases; D043244:Ribonuclease III; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase