A tri-serine cluster within the topoisomerase IIα-interaction domain of the BLM helicase is required for regulating chromosome breakage in human cells.

The recQ-like helicase BLM interacts directly with topoisomerase IIα to regulate chromosome breakage in human cells. We demonstrate that a phosphosite tri-serine cluster (S577/S579/S580) within the BLM topoisomerase IIα-interaction region is required for this function. Enzymatic activities of BLM and topoisomerase IIα are reciprocally stimulated in vitro by ten-fold for topoisomerase IIα decatenation/relaxation activity and three-fold for BLM unwinding of forked DNA duplex substrates. A BLM transgene encoding alanine substitutions of the tri-serine cluster in BLM-/- transfected cells increases micronuclei, DNA double strand breaks and anaphase ultra-fine bridges (UFBs), and decreases cellular co-localization of BLM with topoisomerase IIα. In vitro, these substitutions significantly reduce the topoisomerase IIα-mediated stimulation of BLM unwinding of forked DNA duplexes. Substitution of the tri-serine cluster with aspartic acids to mimic serine phosphorylation reverses these effects in vitro and in vivo. Our findings implicate the modification of this BLM tri-serine cluster in regulating chromosomal stability.

Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells.

Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom's syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.