Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

Abstract

Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' ± 5' DNA helicase identi®ed as a member of the RecQ family. We developed polyclonal antibodies speci®c for the NH 2 -and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/ M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modi®cations in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process.

ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation

“…Furthermore, if BLM phosphorylation was a necessary condition to eciently promote the reverse branch migration of Hollyday junctions, and because BLM phosphorylation requires a functional ATM protein, one would have expected an increased level of SCE in ATM-de®cient cells, which is not the case (Bartram et al, 1976;Galloway, 1977). Finally, our experiments did not allow us to detect any phosphorylated form of BLM in untreated interphase cells, including S phase sorted cells (the present study and Dutertre et al, 2000). For these reasons, we propose that ATM-dependent BLM phosphorylation is involved in another function.…”

“…This partial defect was more pronounced in BS cells lacking BLM expression (GM03403D; Dutertre et al, 2000) than in BS cells expressing a full-length mutated protein (IPM1; Barakat et al, 2000), suggesting that BLM may participate in the G2/M checkpoint through interaction(s) with protein(s). BRCA1, which co-immunoprecipitates with BLM (Wang et al, 2000a), is a good candidate for such an interaction since it has recently been shown that BRCA1 exon 11 isoform-de®cient cells exhibit a defective G2/M cell cycle checkpoint in response to ionizing radiation, suggesting a major role of BRCA1 in maintaining genetic stability, at least in part through the G2/M checkpoint (Xu et al, 1999).…”

Monopolar Spindle 1 (MPS1) Protein-dependent Phosphorylation of RecQ-mediated Genome Instability Protein 2 (RMI2) at Serine 112 Is Essential for BLM-Topo III α-RMI1-RMI2 (BTR) Protein Complex Function upon Spindle Assembly Checkpoint (SAC) Activation during Mitosis

“…4C) were fractionated and analyzed. Fractionation confirmed that BLM and RMI2 were normally present in the nucleoplasmic, nuclear matrix, and chromatin fractions (11) and that hydroxyurea treatment caused BLM to associate with chromatin in all cells tested. Furthermore, cells coexpressing the shRNA and either the recombinant WT or mutant RM12 showed no appreciable difference (Fig.…”

“…taxol and nocodazole) (5,6,8,11). Our group reported earlier that, like BLM, both the endogenous and the exogenously expressed RMI2 is phosphorylated when cells were treated with these agents (5).…”

DEK over-expression promotes mitotic defects and micronucleus formation

“…In fact, the dynamic pattern of DEK localization in mitosis resembles that of many proteins involved in DNA damage repair which are removed from chromatin during mitosis such as Rad51, DNA-PK, FAND2 and Bloom complex components. [62][63][64][65] It is important to point out that while the majority of DEK is absent from mitotic chromosomes until telophase, a greater proportion of the fusion proteins, and particularly the C-terminal DEK-GFP fusion, were in fact prominent on mitotic chromosomes. This increased retention is likely due to the overexpression of DEK.…”