Abstract
Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G2/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.
Highlights
HPso4 has been implicated in the resistance to interstrand cross-linking DNA-damaging agents
In accord with HR being the major pathway that repairs replication-associated double strand breaks (DSBs), we demonstrate a role for the hPso4 complex in this repair pathway, an association with the breast cancer type 1 susceptibility protein (BRCA1) A complex, and a requirement for generating single stranded DNA that is a prerequisite for HR to proceed
We were unable to obtain viable clones of hPso4 knockdown cells in clonogenic survival assays. To determine whether this growth defect was due to the direct involvement of hPso4 in cell proliferation, we examined the subcellular localization of hPso4 and found that it co-localized with the DNA replication clamp proliferative cell nuclear antigen (PCNA) in both unperturbed cells and in cells treated with HU (Fig. 1, C–E), suggesting that hPso4 may directly participate in DNA replication
Summary
HPso4 has been implicated in the resistance to interstrand cross-linking DNA-damaging agents. Depletion of hPso4 or CDC5L in either U20S (Fig. 5, B, C, and G) or HeLa cells (Fig. 5H) resulted in a significant decrease in the number of GFPpositive cells after expression of I-SceI, albeit not to the same extent as when the levels of the key HR proteins, BRCA2 and Rad51, were reduced by siRNA (Fig. 5, D, E, and G).
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