Evidence for BLM and Topoisomerase IIIalpha interaction in genomic stability.
The genomic instability of persons with Bloom's syndrome (BS) features particularly an increased number of sister-chromatid exchanges (SCEs). The primary cause of the genomic instability is mutation at BLM, which encodes a DNA helicase of the RecQ family. BLM interacts with Topoisomerase IIIalpha (Topo IIIalpha), and both BLM and Topo IIIalpha localize to the nuclear organelles referred to as the promyelocytic leukemia protein (PML) nuclear bodies. In this study we show, by analysis of cells that express various deletion constructs of green fluorescent protein (GFP)-tagged BLM, that the first 133 amino acids of BLM are necessary and sufficient for interaction between Topo IIIalpha and BLM. The Topo IIIalpha-interaction domain of BLM is not required for BLM's localization to the PML nuclear bodies; in contrast, Topo IIIalpha is recruited to the PML nuclear bodies via its interaction with BLM. Expression of a full-length BLM (amino acids 1-1417) in BS cells can correct their high SCEs to normal levels, whereas expression of a BLM fragment that lacks the Topo IIIalpha interaction domain (amino acids 133-1417) results in intermediate SCE levels. The deficiency of amino acids 133-1417 in the reduction of SCEs was not explained by a defect in DNA helicase activity, because immunoprecipitated 133-1417 protein had 4-fold higher activity than GFP-BLM. The data implicate the BLM-Topo IIIalpha complex in the regulation of recombination in somatic cells.
Hu
P
P
Laboratory of Cancer Susceptibility, Department of Human Genetics and the Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Beresten
S F
SF
van Brabant
A J
AJ
Ye
T Z
TZ
Pandolfi
P P
PP
Johnson
F B
FB
Guarente
L
L
Ellis
N A
NA
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001816:Bloom Syndrome / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D002461:Cell Line, Transformed; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D010641:Phenotype; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D012854:Sister Chromatid Exchange; D014407:Tumor Cells, Cultured
D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I