Bloom’s syndrome (BS) is a genetic instability disorder resulting from loss of function of the BLM helicase. It confers a marked predisposition to most types of cancer. BS cells are characterized by excessive inter- and intra-chromosomal exchanges as well as chromosomal aberrations highlighting the important role of BLM in maintaining DNA fidelity. BLM unwinds a variety of nucleic acid structures and catalyzes branch migration, single-strand annealing and double Holliday junction dissolution. BLM interacts with proteins involved in DNA damage recognition, signaling and repair. The biochemical activities and protein interactions of BLM are important for various aspects of nucleic acid metabolism including double-strand break repair, DNA replication repair, telomere maintenance and rDNA transcription.
BLM undergoes post-translational modification to coordinate its various activities. This study investigates the effect of one of these post-translational modifications, phosphorylation, on BLM function. Helicase assays were performed with in vitro expressed and purified BLM proteins to show that BLM phosphorylation inhibits the specific activity of BLM. Putative BLM phosphorylation sites were identified using bioinformatics prediction programs. Evolutionary conservation, secondary structure and phospho-motif analyses were completed to identify putative BLM phosphorylation sites. Site-directed mutagenesis generated phosphomimetic and alanine replacement constructs for sixteen of twenty-two putative phosphorylation sites for use in cell biology and biochemical screens. Eight phosphorylation sites were identified that may regulate BLM helicase activity in vitro. Phosphorylation of some BLM residues was validated using mass spectrometry.
The effect of BLM phosphorylation on the replication fork-regression activity of BLM was assessed to determine whether phosphorylation differentially regulates the function of BLM. These studies were carried out in collaboration with Dr. David Orren at the University of Kentucky and identified five phosphorylation sites that may regulate the replication fork-regression activity of BLM. Subsequent analyses focused on S1290 and S1296; the combination of cellular fractionation and phosphorylation-specific antibodies indicated that S1290 and S1296 are phosphorylated after replication stress and in mitosis. Immunofluorescence studies suggested that modification of these residues may control the localization of BLM to sites of stalled replication.
Interactions between BLM and topoisomerase IIα are required to prevent chromosome breaks. Interactions peak in mitosis when BLM is highly phosphorylated suggesting that BLM phosphorylation may regulate or fine-tune these interactions. The topoisomerase IIα-interaction region of BLM is required for topoisomerase IIα stimulation of BLM helicase activity; helicase assays using phosphatased BLM proteins demonstrate that BLM phosphorylation is similarly required for topoisomerase IIα-mediated stimulation. Therefore, we tested the hypothesis that phosphorylation of specific residues in the topoisomerase IIα interaction region of BLM regulate the interaction between BLM and topoisomerase IIα. We examined the effects of phosphorylation on three residues in the topoisomerase interaction region of BLM using helicase assays and protein interaction studies, although studies were inconclusive.