Escherichia coli RecG functionally suppresses human Bloom syndrome phenotypes
© Killen et al.; licensee BioMed Central Ltd. 2012
Received: 21 January 2012
Accepted: 11 October 2012
Published: 30 October 2012
Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.
Human cells possess five proteins with clear sequence homology to the E. coli RecQ protein: BLM, WRN, RECQL, RECQL4 and RECQL5. These proteins are all implicated in preserving genomic integrity (reviewed in[1, 2]). Functionally, inherited homozygous defects in BLM, WRN or RECQL4 cause human disease: Bloom syndrome, Werner syndrome and Rothmund-Thomson/RAPADILINO/Baller-Gerold syndromes respectively. Bloom syndrome is particularly striking for its predisposition to early-onset malignancy with a broad distribution of cancer types similar to that seen with sporadic tumors in the general population.
Sequence homology of BLM with RecQ notwithstanding, characterization of the in vitro activities of BLM demonstrates significant similarities to the biochemistry of the E. coli RecG DNA translocase protein. Both BLM[4, 5] and RecG[6, 7] can bind to and regress multi-stranded DNA structures that model stalled replication forks. Similarly, both BLM[8, 9] and RecG[10, 11] have the capacity to bind to and branch migrate Holliday junctions. Both BLM[12, 13] and RecG have also been shown to dismantle D-loops where a 3′-OH ssDNA has invaded a homologous DNA duplex, although the mechanism by which RecG carries out this reaction is less well established. The manner by which RecG accomplishes these tasks is in large part made clear by its crystal structure: a RecG monomer binds at a model replication fork by inserting a C-terminal protein wedge domain into the fork. The body of RecG then functions as a double stranded DNA translocase to pull on and reanneal DNA template strands through the body of the protein. At such time as the nascent DNA strands encounter the wedge domain, they are stripped off and allowed to anneal together resulting in the formation of a Holliday junction. As RecG continues to translocate on the dsDNA, the branch point of the Holliday junction is effectively migrated. In the absence of high-resolution structural information it remains unclear precisely how BLM carries out these activities.
The BLM protein also possesses activities it is not known to share with RecG. BLM can act in concert with EXO1 at double stranded DNA ends to cause a 5′-3′ single stranded resection that exposes a free ssDNA 3′ end suitable for loading with Rad51, reminiscent of the combined activities of the E. coli RecQ helicase and RecJ 5′-3′ exonuclease[18, 19]. Alternatively, BLM can also functionally interact with the DNA2 exonuclease to carry out a similar reaction. BLM has strong unwinding activity on G-quadruplex DNA structures as well as both ssDNA annealing[22, 23] and/or strand exchange activities. Notably, BLM has many well-characterized protein-protein interactions, including those with RMI1, C16orf75 (RMI2) and TOP3A[25–28] that collectively mediate double-Holliday junction dissolution, as well as direct interaction with the Rad51 recombinase and with the multi-component Fanconi anemia protein containing BRAFT complex. In contrast, RecG functions in a largely monomeric manner.
The mechanistic similarities between BLM and RecG have led us and others to speculate that E. coli RecG and human BLM may be functional analogs. In order to test this hypothesis and to determine the extent to which the shared biochemical activities of BLM and RecG are responsible for suppressing the functional cellular phenotypes observed in human cells lacking BLM, we reasoned that suitable expression of RecG might suppress a BLM defect. The best characterized cellular phenotype of BLM deficiency is a 10-fold elevated frequency of sister chromatid exchanges, thought to represent a hyper-recombination phenotype indicative of elevated crossing-over and overall genomic instability. In addition, we have recently demonstrated that BLM deficiency causes a striking destabilization of the highly repetitive human ribosomal RNA gene clusters (the ‘rDNA’), with recombination-mediated genomic restructuring of these clusters increased 100-fold over cells wild-type for BLM function. Accordingly, we engineered several semi-humanized RecG protein expression systems and stably introduced these constructs into human cells either wild-type or defective for the BLM protein. We then assayed the resulting RecG transgene expressing cells for changes in these two phenotypes.
Protein expression constructs
Cell lines used that express normal BLM include the SV40-transformed fibroblast line GM00637 (Coriell Cell Repositories) “BLM+” and the cervical carcinoma line HeLa S3 (ATCC: American Type Culture Collection) “HeLa”. Cell lines used that are deficient in BLM protein include GM08505 (Coriell Cell Repositories) which are SV40 transformed fibroblasts derived from a Bloom syndrome patient homozygous for the Ashkenazi Jewish founder BLM mutation (6-bp del/7-bp ins) at nucleotide 2281 of the open reading frame, hereafter referred to as “BLM-”. A second BLM deficient line used was GM16375 (Coriell Cell Repositories) which are EBV transformed lymphocytes from a French-Canadian Bloom syndrome patient homozygous for a C>A transversion resulting in a (S595X) termination mutation, hereafter referred to as “BLM- FC”. The BLM-defective line stably suppressed by either BLM cDNA expression, or by a control empty vector are the lines PSNF5 “BLM-: cDNA” and PSNG13 “BLM-: empty vector” respectively, as described in (kind gift from Ian Hickson) both derived originally from the GM08505 line (Coriell Cell Repositories). Lines were generally grown in MEM with 10% fetal bovine serum, with L-gln and antibiotic supplementation at 37C in a humidified 5% CO2 incubator.
Stable cell lines were generated by electroporating either a RecG expression construct or a control EGFP expression construct into cells, followed by unselected cell population expansion, one round of flow-sorting enrichment for green fluorescent cells, further unselected expansion, and finally a second round of flow-sorting enrichment for green fluorescent cells. Clonal and subclonal derivatives of these highly enriched fluorescent populations were subsequently derived by limiting dilution. All of the transgene expressing cell lines generated and used in this work are either clonal or subclonal, with the exception of the “BLM- FC: RecG fuse” line: BLM- FC cells were transduced by a high-titer lentivirus (Welgen, Inc.) containing an expression cassette for the RecG-EGFP fusion construct (Figure1A) and separated into green fluorescent and non-fluorescent populations by flow sorting, and the BLM-: EGFP line which was isolated on the basis of drug resistance only. Stable cell lines were chosen for further experiments on the basis of the levels of EGFP expressed in these lines, as measured by flow cytometry.
Sister chromatid exchange assays
Sister chromatid exchange assays were performed largely according to with minor modifications. Cellular metaphase spreads were imaged and scored individually by counting the number of visible exchanges and the number of chromosomes in each unique high powered microscope field to calculate the number of SCEs per chromosome. In our hands, scoring the number of SCEs per chromosome is more robust than scoring the number of SCEs per metaphase and is relatively insensitive to the ploidy of the cell line under investigation. The resulting SCEs/chromosome figures were binned and plotted. All statistical tests were performed using unbinned data. SCE experiments were generally performed by collecting fixed metaphase cells from one or two experiments, which were subsequently dropped on microscope slides to release the metaphase spreads, stained and counted together in a single session at the microscope.
Protein extracts were prepared using RIPA buffer as described previously. All resolving SDS-PAGE gels used 9% acrylamide and were blotted onto Hybond-ECL nitrocellulose membrane (Amersham Biosciences, cat. #RPN68D). Primary antibodies used were: rabbit anti-GFP (Cell Signaling Technologies, cat. #2555), rabbit anti-beta-actin (Cell Signaling Technologies, cat. #4970), rabbit anti-beta-tubulin (NeoMarkers, cat. #RB-9249-PO). The secondary antibody was ImmunoPure Antibody donkey anti-rabbit IgG conjugated with horseradish peroxidase (Pierce, cat. #31458). Blots were developed using an Amersham™ ECL Plus western blotting detection system (GE Healthcare, cat. #RPN2132) and imaged with a Storm 860 PhosphorImager (Molecular Dynamics).
Gene cluster instability analysis was carried out as described previously. Briefly, genomic DNA was prepared in the solid phase by digesting single cell suspensions in agarose with proteinase K in the presence of sarkosyl and EDTA, rinsed thoroughly and equilibrated in 50% glycerol/10 mM Tris/1 mM EDTA pH 8.0 and stored at -20C. 10 μl agarose slices containing approximately 1 μg genomic DNA were equilibrated in suitable restriction digestion buffer and digested overnight with EcoRV (New England Biolabs). Digested DNA still in solid form was loaded into a 1% PFC agarose (Bio-Rad) gel and run in 0.5× TBE buffer (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA pH 8.0) using a CHEF-MAPPER system (Bio-Rad) at 14C. Pulsed-field electrophoretic conditions were a field strength of 6 V/cm with 120° separation between field vectors. Field switch times varied from 3 seconds to 90 seconds with a ‘ramp factor’ of 0.357. Gels were run for 24 hours, equilibrated to 0.5% glycerol in water, then dried, rehydrated, probed with a radiolabeled probe specific for the human rDNA and imaged with a Storm 860 PhosphorImager (Molecular Dynamics).
EGFP quantitation by flow cytometry
For cell lines carrying EGFP transgenes, the fold increase in fluorescence relative to non-fluorescent cells was calculated by dividing the geometric mean value of green fluorescence emitted from the fluorescent cell sub-population by the geometric mean value of background green autofluorescence from the non-fluorescent sub-population of the same cell line.
RecG expression suppresses the BLM- elevated SCE phenotype
SCE levels and EGFP expression
HeLa: RecG fuse
BLM+: RecG 2a
BLM-: RecG 2a high
BLM-: RecG fuse high
BLM-: RecG 2a low
BLM-: empty vector
BLM- FC: RecG fuse
BLM-: RecG fuse med
BLM-: lost RecG fuse
RecG has no effect on SCE levels in cells expressing normal BLM protein
Loss of RecG expression restores the elevated SCE phenotype to BLM- cells
RecG expression reduces the elevated gene cluster instability (GCI) of BLM- cells
RecG expression does not affect gene cluster instability in BLM+ cells
The E. coli RecG protein, with essentially no sequence similarity to human BLM, would seem unlikely to engage in any of the well-characterized protein-protein interactions that are important for function of the BLM protein in the wild-type human cellular context, although we have not experimentally ruled out these interactions. The capacity with which RecG can suppress BLM- cellular phenotypes suggests therefore that the physiological role of BLM in suppressing the two phenotype we investigated, sister chromatid exchanges and gene cluster instability, is to perform the same molecular reactions that can be performed in a human cell by the RecG monomer alone, namely direct manipulations of DNA structures. This is not to rule out a role for BLM and associated protein-protein interactions in suppression of other manifestations of genomic instability, or potentially BLM accessory proteins may help localize BLM to DNA structures upon which both BLM and RecG can act, thereby reducing the required level of BLM in the cell relative to the large amount of ectopic RecG expressed in the cell lines of this study. Activities of BLM not known to be shared by RecG, such as the unwinding of G-quadruplex DNA, must play a minor role in both sister chromatid exchange and gene cluster instability suppression assayed herein, or alternatively, a G-quadruplex activity of RecG may remain to be discovered.
We suggest that RecG and BLM fulfill the same physiological function in E. coli and human cells respectively: they recognize stalled/regressed DNA replication forks and then utilize their common biochemical functions to mitigate this topological genomic damage. Given the well-established biochemical similarities between the two proteins and the capacity for RecG to functionally substitute for BLM shown in this work, we consider this the most parsimonious conclusion. Nevertheless, an alternative possibility consistent with our data is that the lesions recognized by BLM and RecG may be distinct from each other, but that may be converted though subsequent processing into a common form. For example, human cells, lacking RecG, may over-produce the classic substrate for RecG: a replication fork missing the nascent leading strand which may then be converted, by some as yet uncharacterized mechanism, into a bona fide distinct substrate for the BLM protein. The converse situation is also possible: that in the absence of BLM, the lesion recognized by BLM is converted into a different lesion that can be recognized and repaired by RecG. An additional possibility is that the lesion recognized by BLM and RecG may be the same, yet the two proteins may convert the lesion to different, but still less genomically destabilizing alternative structures. Mechanistic details aside, that the bacterial RecG protein can relieve human genomic instability phenotypes resulting from BLM protein deficiency demonstrates the highly conserved nature of DNA structural lesions and the type of mitigating enzymatic processing required to preserve genomic stability.
The role of BLM in human genomic stabilization is well-established. In contrast, a major mechanistic role of RecG in E. coli genomic stabilization has only recently been elucidated. In the absence of RecG, PriA initiates spurious replication forks throughout the bacterial genome, causing poorly-controlled genomic over-replication and compromised viability. In the absence of RecG, cells have an absolute requirement for a ssDNA exonuclease activity, unless PriA is also eliminated. It will be interesting to determine whether human BLM- cells share this over-replicated phenotype. Curiously, the E. coli RecQ protein rather than protecting the cell from aberrant recombination structures as the human BLM and E. coli RecG proteins do, seems to act instead to promote their formation. Cells lacking the UvrD-mediated inhibition of recombination and also lacking RecG are killed by formation of intermolecular recombination intermediates (IRIs) that interfere with correct genome segregation to daughter cells. Formation of IRIs is caused by the action of RecQ and partner proteins and causes “death by recombination”. Deletion of RecQ restores cellular viability. An analogous phenotype in human cells might be the elevated formation of anaphase bridges in cells deficient for BLM. One evolutionary interpretation consistent with our functional data here would be that although the ancient common protein ancestor of both RecQ and BLM was preserved in evolutionary descent through both lineages to provide a core helicase domain, the functionality of these proteins diverged in opposite directions. Since there are no RecG homologs by sequence similarity in human cells, it would appear that at least one of the five human RecQ paralogs, BLM, has at least to some extent taken on the important recombination molecular transactions provided to E. coli by the RecG protein, and is an example of convergent evolution.
E. coli RecG and human BLM play similar, and to some extent interchangeable, roles in suppressing genomic instability phenotypes when expressed in human cells. Accordingly, given their lack of sequence homology, E. coli RecG and human BLM may be an example of convergent evolution.
The authors very gratefully acknowledge the assistance of Dr. Bob Lloyd (University of Nottingham) for assessing the functionality of the RecG-EGFP fusion protein in E. coli. This work was supported by the Markey Cancer Research Center.
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