| DNA helicases play essential roles in DNA replication, repair and recombination pathways. DNA helicase B has been identified in two mammalian species, mouse and human. The human DNA helicase B (HDHB) cDNA was cloned by homology to the mouse gene. The predicted protein is 1087 residues in length and contains conserved motifs characteristic of helicase superfamily 1, which are strikingly similar to those of E. coli RecD and T4 Dda proteins.; Previously, we have found that a dominant negative point mutant HDHB (MutB) could block DNA replication. To analyze the subcellular localization of wt HDHB, GFP-HDHB fusion proteins were transiently expressed. In G1 phase cells, the fusion protein localized in DNA damage nuclear foci, while in S phase cells it localized in the cytoplasm, suggesting the existence of cell cycle-dependent localization signal(s) in the protein. Mutational analysis suggests that there is a functional subcellular localization domain in the extreme C-terminus of HDHB. We have also found that HDHB is phosphorylated in vivo in a cell cycle-dependent pattern, and that phosphorylation of ser 967 regulates the subcellular localization of the protein. In vitro, cyclin dependent kinase (CDK) phosphorylated purified recombinant HDHB on the same sites phosphorylated in vivo. Consistent with this, we found the subcellular localization of endogenous HDHB is also cell cycle-dependent.; Tagged HDHB co-localizes in nuclear foci with endogenous Mre11, NBS1, Rad50, ATM, and RPA, and co-immunoprecipitates with Mre11 and ATM. Cells expressing GFP-HDHB, but not helicase-deficient HDHB, contain single-stranded DNA (ssDNA) in vivo and pan-nuclear gammaH2AX. These observations suggested that unregulated processing of endogenous DNA damage by over-expressed HDHB promotes damage signaling.; To examine whether endogenous HDHB participates in damage signaling, stable HDHB-knockdown cell lines were generated. HDHB-depleted cells are sensitive to ionizing radiation and display a significant delay in radiation-induced phosphorylation of NBS1, H2AX, and Chk2, particularly in G1 phase. NBS1 and RPA focus formation after irradiation is also delayed in HDHB-depleted cells. We suggest that endogenous HDHB associates with MRN complexes at sites of damage, generating ssDNA that amplifies damage signals. |
