title: Proteolysis of the human DNA polymerase delta smallest subunit p12 by μ-calpain in calcium-triggered apoptotic HeLa cells.
abstract: Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Fan
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: You
authors.affiliation: School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Yuanxia
authors.initials: Y
authors.lastname: Qian
authors.affiliation: School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Liu
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Huiqing
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Huifang
authors.initials: H
authors.lastname: Song
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Keping
authors.initials: K
authors.lastname: Chen
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, People's Republic of China.
authors.forename: Yajing
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics*; D059748:Proteolysis
chemical_list: C521101:POLD4 protein, human; D004258:DNA Polymerase III; D002154:Calpain; C449002:mu-calpain; D002118:Calcium