Roles of DNA polymerase eta and replication protein A (RPA) in undamaged and platinum-treated human cells

Platinum-based drugs are widely used in cancer therapy. Bypass of platinum-induced DNA adducts during DNA replication by specialised DNA polymerases may contribute to drug tolerance and tumour cell resistance. Human cells lacking the POLH gene product, DNA polymerase eta (pol eta), are characterized by increased sensitivity to cisplatin and related platinum-based chemotherapeutic drugs. To directly investigate the role of pol eta in bypass of platinum-induced lesions during DNA replication in vivo, DNA combing was used to compare the length of individual nascent DNA strands in pol eta-deficient XP30RO cells and in a derivative cell line, TR30-2, in which wild-type pol eta is expressed from a POLH transgene. Following treatment with cisplatin or carboplatin, nascent DNA strands were on average up to 39% shorter in cells lacking pol eta than in cells expressing pol eta, consistent with a role for pol eta in bypass of platinum-induced lesions during DNA replication in vivo. This provides direct evidence at the level of individual DNA strands that pol eta modulates nascent strand length in human cells exposed to platinum-based chemotherapeutic drugs. Consistent with checkpoint activation, cisplatin and carboplatin induced phosphorylation of Chk1 on serine 317. Moreover, damage-dependent phosphorylation of histone 2AX on serine 139 was significantly increased in cells lacking pol eta, indicating enhanced DNA damage signalling. Phosphorylation of replication protein A (RPA), the main ssDNA binding protein was also investigated. RPA, a heterotrimeric protein, involved in most aspects of normal DNA metabolism including replication, recombination and repair, is regulated by phosphorylation of the N-terminal domain of the 34kDa RPA2 subunit. Phosphorylation is carried out in a cell-cycle-dependent manner by cyclin-dependent kinases, and following DNA damage by PIK kinases. Using EdU labelling and immunofluorescence, cisplatin- and carboplatin-induced phosphorylation of RPA2 on Ser4/Ser8 was directly correlated with the severity of replication inhibition in individual, pol eta-deficient cells. This data provides novel insights into the role of pol eta and RPA in DNA replication and the DNA damage response in human cells following treatment with platinum-based chemotherapeutic drugs. Phosphorylation of Ser4/Ser8 of RPA2 is associated with induction of DNA damage. Further investigation of RPA2 phosphorylation revealed that phosphorylation on Ser4/Ser8 could also be detected in mitotic cells. Durig mitosis the majority of the RPA2 protein is excluded from the chromatin. However, using the phospho-Ser4/Ser8 RPA2 specific antibody in immunofluorescence it was found that during normal mitosis, a fraction of RPA2 protein localised to the centrosome, near the centromere and to the midbody at various mitotic stages. RPA2 is dephosphorylated during late cytokinesis, before cells enter the G1-phase. These data may indicate a novel role for RPA in mitosis and cytokinesis in human cells.

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