BLM also associates with several telomere-specific proteins, such as POT1, TRF1 and TRF2 [34]C[37]. antibodies to BRCA1 (green), BLM (white) or PML (top: white; bottom: green), telomeres were labeled by FISH with a PNA probe (red), and nuclei were stained with DAPI (blue). Yellow arrows indicate foci with all three signals.(TIFF) pone.0103819.s002.tif (1.6M) GUID:?EB56333E-3965-4733-8158-32375774F44D Data Availability StatementThe authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Abstract 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 Chrysophanic acid (Chrysophanol) 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 Chrysophanic acid (Chrysophanol) 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. Introduction Telomeres are DNA-protein complexes comprised of repetitive non-coding DNA sequences at the ends of eukaryotic chromosomes and the proteins that bind these sequences. In mammals, telomeres consist primarily of TTAGGG sequences [1]C[5]. Telomeres prevent chromosome erosion and loss of coding sequences due to the end-replication problem. Loss of telomeric DNA is linked with cellular senescence and aging, and likely resembles double-strand breaks that activate DNA damage response pathways [6]C[9]. While cell growth continuously reduces telomere length, cancer cells become immortalized by activating mechanisms of telomere maintenance. The most common mechanism is expression of the enzyme telomerase, which catalyzes the addition of repeats to maintain telomere length. Approximately 15% of human tumors maintain telomeres independently of telomerase and use a recombination-based mechanism known as alternative lengthening of telomeres (ALT) to maintain telomere lengths [10]C[17]. ALT cells are typified by the presence of ALT-associated PML bodies (APBs) that include telomeric DNA and telomeric proteins [15], [18]. Although the functions of APBs are unclear, they are considered primary sites of telomere metabolism. Aberrant telomere metabolism results in telomere dysfunction, yield chromosomal abnormalities, such as chromosome end-to-end fusions, telomeric translocations, tri- Rabbit Polyclonal to Cytochrome P450 17A1 and quadri-radial chromosomes, and limit growth potential [8], [19]C[22]. The mechanisms of ALT remain unclear. However, several DNA damage response proteins are implicated in ALT due to their association with telomeres or APBs, including the recQ-like helicases BLM (defective in Bloom’s syndrome) and WRN (defective in Werner’s syndrome), Chrysophanic acid (Chrysophanol) and the tumor suppressor BRCA1 [23]C[30]. BLM inhibits recombination by facilitating the resolution of replication and recombination intermediates. Through its structure-specific unwinding activity, BLM really helps to solve DNA damage-induced replication obstructs that if still left unresolved can lead to aberrant recombination and chromosomal damage. BLM affiliates with many proteins involved with DNA repair which includes BRCA1, DNA topoisomerases, DNA mismatch restoration Fanconi and proteins anemia proteins, and is an element from the BRCA1-linked genome surveillance complicated (BASC) [31]C[33]. BLM affiliates with many telomere-specific protein also, such as for example Container1, TRF1 and TRF2 [34]C[37]. Biochemically, Container1 stimulates BLM unwinding of telomeric DNA end structures including G-quadruplexes and D-loops during DNA replication and/or recombination. TRF1 and TRF2 modulate BLM function using telomeric substrates also. The function of BLM in telomere metabolic process is certainly emphasized by telomere dysfunction in cellular material from people that have Bloom’s symptoms.
