This work documents assays for the function of the normal BLM
cDNA in human cells, in yeast cells, and in enzymatic assays. Transfection of the normal BLM
cDNA reduces the high SCE phenotype of BS cells. Similar results have been observed previously (Giesler et al., 1997
). Transfection of the normal cDNA restores BLM to the nucleus of BS cells. BLM has enzymatic properties in vitro consistent with its predicted membership in the RecQ family of DNA helicases. Similar results have been reported previously (Karow et al., 1997
). Two missense alleles of BLM
found in individuals with clinical BS (Ellis et al., 1995a
) encode full-length BLM protein that lacks helicase displacement activity in vitro and fails to reduce the high-SCE phenotype of BS cells, as does a constructed missense mutation disrupting the helicase ATP-binding site. In S. cerevisiae
the expression of the normal BLM
cDNA from a strong inducible promoter can complement the growth phenotype of an sgs1 top3
strain but not that of an sgs1 top1
strain. In sgs1 top3
cells, expression of Sgs1p or BLM creates an HU-sensitive growth phenotype. This phenotype is dependent on expression of a helicase-competent BLM
Normal and missense proteins are recovered from AMR61 cells as stable, soluble proteins in approximately the same yield. This suggests that the missense proteins fold close to the normal conformation because yeast cells do not degrade them differentially relative to normal or package them into an insoluble form. The Q673R missense protein has 37% of the DNA-dependent ATPase specific activity of the normal protein, demonstrating that it retains some normal function and therefore must be close to normal conformation.
Antibodies raised against the N-terminal region of the BLM cDNA sequence are used here to demonstrate the nuclear localization of BLM, its focal arrangement in the nucleus, and its absence from cells derived from persons with BS. This reagent allows the evaluation of BS cells transfected with different BLM
alleles. The focal organization of BLM is restored to BS cells by transfection of the normal BLM cDNA. Missense alleles in BLM are expressed in variable amounts in the different stable transfected cell lines and fail to localize in the numerous bright, discrete nuclear foci that are seen by immunofluorescence analysis of normal human cells. The significance of this focal pattern of localization of BLM in the nucleus of human cells remains to be determined. The lack of extensive amino acid identities between the N- and C-terminal domains of BLM and WRN, and the profound differences between the clinical phenotypes of the affected individuals (German, 1993
; Epstein et al., 1996
), suggest specialized roles or different cellular locations for these two helicases. Recently WRN has been shown to be present in the nucleolus of human cells, a location distinct from the major sites of BLM localization (Marciniak et al., 1998
), a finding consistent with this hypothesis.
Differences were seen among cloned cell lines derived from HG2522 cells transfected with the three different missense alleles. These cell lines express BLM genes from a strong constitutive promoter (RSV) and were selected for good growth in culture. The plasmids encoding the two missense alleles found in BS individuals, Q672R [139(ViKr)] and C1055S [113(DaDe)], transfected nearly as well as the normal BLM cDNA. The three missense alleles of BLM studied here alter amino acids that are either conserved in all family members (Q672R and K695T) or in the extended C-terminal homology domain (C1055S). The C1055S missense allele showed stable accumulation of full-length BLM, but the protein was present in a diffuse overall nuclear staining pattern. The cells transfected with the Q672R missense allele showed a diffuse pattern and few small dots. These cloned cell lines expressing the Q672R and C1055S missense proteins grew fairly well and generated multiple stable cell lines (six of six). In contrast, most of the cell lines transfected with the helicase domain knockout allele (K695T) died in culture (five of six and three of six in two independent transfection experiments) and express very little stable protein. The K695T mutation may potentially function as a dominant negative mutation. What BLM is present in these surviving cells appears to be in a few small dots per nucleus or diffusely localized. These observations suggest that the Q672R and K695T missense proteins can assemble into a small number of focal nuclear structures but fail to form as many nuclear foci as the normal BLM gene product does.
Because the missense proteins are found in lower amounts in these stable transfected cell lines relative to normal BLM, the failure to reduce the high SCE phenotype may be due simply to the lower concentration of these proteins in the selected cell lines rather than the loss of BLM function. Another factor in this analysis is the stable focal localization of the normal protein and the generally diffuse pattern seen with the missense proteins. BLM may function in these nuclear foci, and the failure of the missense proteins to localize into or form these numerous structures may be the reason for their failure to reduce the SCEs. The missense alleles may accumulate in HG2522 cells to a lesser extent than normal BLM and fail to be incorporated into nuclear foci because they are not recognized by a protein partner because they are not folded properly. Misfolding of the missense proteins would create an unstable molecule that would likely be targeted for proteolysis; however, the proteins are expressed in yeast cells as soluble proteins to approximately the same yield, and the Q672R missense protein retains some enzymatic activity in vitro. This indicates that the overall structure of these proteins is likely to be close to normal. They may fail to be localized focally and accumulate to the same stable concentration as normal BLM if the incorporation of BLM molecules that are inactive but of normal conformation may form poisonous complexes that are dispersed or unstable in the nucleus. The K695T missense protein is especially deleterious to the cells used in this study. This missense gene was constructed in vitro, whereas the other two missense alleles are found in affected individuals, consistent with the transfection efficiency in vitro. The stability of the mutant proteins in yeast cells, the immunofluorescent results, and the transfection data support the idea that the activity and location of the missense BLM proteins and not simply the lower concentrations of these proteins are the essential features of the failure to reduce the SCEs in BS cells.
DNA helicases, like topoisomerases and other enzymes that manipulate DNA strands, can be disruptive if unregulated. The activity and localization of these enzymes must be controlled to prevent collisions with polymerases and alterations of DNA topology that might disrupt gene expression. Other circumstantial evidence that supports the idea that defective RecQ helicase proteins are deleterious to the cell is the fact that the nuclear localization signal of both WRN and BLM is found in the last 100 amino acids of these large proteins, suggesting a cellular safety mechanism such that mutant helicases arising from translational stop signals can never be nuclear-localized (Kaneko et al., 1997
; Matsumoto et al., 1997
In sgs1 top3 S. cerevisiae
cells the HU-sensitive phenotype seen when Sgs1p or BLM is expressed may reflect an enhanced rate of ectopic recombination occurring in these cells because HU depletion of deoxynucleotide triphosphate pools causes stalled and broken replication forks (Vassilev and Russev, 1984
; Kuzminov, 1995
). The stalling of the replication forks can create additional single-stranded regions in cells allowing the entry of these helicases into the DNA duplex, creating additional single-stranded DNA that can invade a neighboring DNA duplex, especially a sister chromatid. The three missense alleles of BLM
that lack in vitro helicase activity do not confer this HU-sensitive phenotype, demonstrating that the helicase activity of BLM is required. These additional recombination events may not be resolved in a timely manner such that cells enter mitosis with entanglements, as is thought to occur in the rqh1-h2
) mutants of S. pombe
(Stewart et al., 1997
). It is possible that S and G2 cell cycle checkpoints that monitor the completion of DNA replication and block mitosis in the presence of DNA damage fail to recognize unresolved recombination junctions between sister chromatids as damage or as potentially deleterious. Failure to resolve these events in BS cells in a timely and efficient manner could lead to an elevated frequency of nondisjunction and somatic mutation by an error-prone repair mechanism.
Other models for the function of this helicase include a role during S/G2 phase to help remove single-stranded DNA created by replication slippage (Schachman et al., 1960
) in AT-rich repeated-sequence elements that may anneal ectopically or a role in unwinding sequence-specific DNA conformations that repeated-sequence elements may assume potentially. An interesting relationship between Sgs1p, WRN, and the nucleolus has been found recently by the Guarente laboratory (Sinclair and Guarente, 1997
; Sinclair, et al., 1997
; Marciniak et al., 1998
), suggesting a role for RecQ DNA helicases in the stability of rDNA repeats. Recently the telomeric regions of the chromosomes of Ustilago maydis
were isolated and found to contain RecQ DNA helicase genes as one of the two major middle repeated subtelomeric sequences (Sanchez-Alonso and Guzman, 1998
), suggesting a need for multiple RecQ helicase genes in this highly recombinogenic fungi.
One of the major characteristics of the few known disorders that feature genomic instability is the potential for somatic mutation and disintegration of the genomic complement at each cell division. Bloom syndrome is one of the most cancer-prone disorders known (German, 1993
). The work reported here demonstrates that the DNA helicase activity of the BLM gene product is important for the maintenance of genomic stability and for the stable localization and function of BLM in complexes in the nucleus of human cells. Therefore the loss of this DNA helicase leads ultimately to the development of cancer in persons with BS, and the elucidation of its structure and function will lead to a new understanding of one mechanism by which neoplastic cells can arise and progress into clinical cancer in normal individuals.