Passage
The Bloom syndrome helicase (BLM) transcript is catalytically inactive in Bloom syndrome (BS) , a genetic disorder that leads to genomic instability characterized by excessive somatic recombination events. Research studies have shown that BLM may be involved in DNA synthesis repair of stalled replication forks during replication stress (arrest) . To investigate this further, an experiment was conducted using cell lines derived from a patient with BS. One set of cells differed only by BLM status: PSNF5 (BLM⁺, vector with BLM cDNA transfected) and PSNG13 (BLM⁻, empty vector transfected) . Additional mutations, BLM-K695T and BLM-T99A, were studied in stable transfected cells.The nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) were used to label and track genomic regions of active replication. Cells were initially treated with IdU for 15 minutes. Subsequently, either 4 mM of hydroxyurea (HU) or 30 µM of aphidicolin (Aph) were used to induce replication stress for 4 hours, after which cells were treated with CldU for 85 minutes to assess replication recovery. Cells were mounted on microscope slides, lysed to isolate chromosome fibers, and then fixed on the slides. Sites of IdU and CldU incorporation into the growing DNA strand were then detected by immunostaining the isolated chromosome fibers with analog-specific, fluorescent-coupled antibodies. Replication activity was visualized via microscopy and measured.
Figure 1 (A) Experimental protocol and (B) fluorescent imaging of a microscope slide showing incorporation of IdU (black lines) and CldU (gray lines) into DNA fibers isolated from a group of BLM⁺ and BLM⁻ cells, with single fibers depicted in each row
Figure 2 Replication fork recovery in cells expressing BLM⁺, BLM⁻, and the mutation BLM-K695T (antagonistic to wild-type)
Figure 3 New sites of replication in cells expressing BLM⁺, BLM⁻, and the mutation BLM-T99A (T99 phosphorylated after replication arrest)
Adapted from Davies SL, North PS, Dart A, Lakin ND, Hickson ID. Mol Cell Biol. 2004;24(3) :1279-91.
-After Aph treatment, replication fork recovery in PSNF5 and PSNG13 cells was analyzed as a function of time. Which of the following conclusions explains the data shown in the graph below?

Question

Passage
The Bloom syndrome helicase (BLM) transcript is catalytically inactive in Bloom syndrome (BS) , a genetic disorder that leads to genomic instability characterized by excessive somatic recombination events. Research studies have shown that BLM may be involved in DNA synthesis repair of stalled replication forks during replication stress (arrest) . To investigate this further, an experiment was conducted using cell lines derived from a patient with BS. One set of cells differed only by BLM status: PSNF5 (BLM⁺, vector with BLM cDNA transfected) and PSNG13 (BLM⁻, empty vector transfected) . Additional mutations, BLM-K695T and BLM-T99A, were studied in stable transfected cells.The nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) were used to label and track genomic regions of active replication. Cells were initially treated with IdU for 15 minutes. Subsequently, either 4 mM of hydroxyurea (HU) or 30 µM of aphidicolin (Aph) were used to induce replication stress for 4 hours, after which cells were treated with CldU for 85 minutes to assess replication recovery. Cells were mounted on microscope slides, lysed to isolate chromosome fibers, and then fixed on the slides. Sites of IdU and CldU incorporation into the growing DNA strand were then detected by immunostaining the isolated chromosome fibers with analog-specific, fluorescent-coupled antibodies. Replication activity was visualized via microscopy and measured.

Figure 1 (A) Experimental protocol and (B) fluorescent imaging of a microscope slide showing incorporation of IdU (black lines) and CldU (gray lines) into DNA fibers isolated from a group of BLM⁺ and BLM⁻ cells, with single fibers depicted in each row

Figure 2 Replication fork recovery in cells expressing BLM⁺, BLM⁻, and the mutation BLM-K695T (antagonistic to wild-type)

Figure 3 New sites of replication in cells expressing BLM⁺, BLM⁻, and the mutation BLM-T99A (T99 phosphorylated after replication arrest)
Adapted from Davies SL, North PS, Dart A, Lakin ND, Hickson ID. Mol Cell Biol. 2004;24(3) :1279-91.
-After Aph treatment, replication fork recovery in PSNF5 and PSNG13 cells was analyzed as a function of time. Which of the following conclusions explains the data shown in the graph below?

Quizplus · Accepted Answer

11ecba2d_0f49_019d_a3bf_75225706a364_MD0008_00 In the experiment, researchers inhibited replication for 4 hours by treating cells with Aph and then analyzed the recovery of DNA synthesis for 85 minutes. The graph shows that even at the 85-minute time point, PSNG13 cells continued to have a lower replication fork recovery rate than PSNF5 cells. Both cell types differ only by BLM expression status, with BLM being active in PSNF5 (BLM⁺) cells but inactive in PSNG13 cells (BLM⁻). Paragraph 1 states that there is an excess of somatic recombination events in the setting of BS, which leads to genomic instability. Therefore, compared to PSNF5 (BLM⁺) cells, a lack of BLM in PSNG13 cells led to increased somatic recombination events (genomic instability) characteristic of BS and also resulted in a lower rate of DNA synthesis (ie, lower replication fork recovery). Accordingly, during the replication recovery fork period, PSNF5 and PSNG13 cells will have differing rates of somatic recombination and DNA synthesis. (Choice A) The passage states that BLM is already absent in PSNG13 cells; therefore, the time when replication fork recovery is assessed does not provide any relevant information about BLM expression. In addition, the time points assessed correspond only to replication fork recovery rates and therefore cannot conclusively predict BLM expression levels. (Choice C) Compared to PSNF5 cells that express BLM, PSNG13 cells are more sensitive (ie, more damaged/affected) to Aph treatment because they completely lack the BLM transcript necessary for adequate replication fork recovery in the setting of replication stress. (Choice D) The time at which replication fork recovery was assessed is the independent variable, which was purposely altered in the experiment to assess its effect on the dependent variable (ie, replication fork recovery percentage). Therefore, the percentage of replication forks that can be rescued depends on recovery time (ie, how long the researchers evaluate fork recovery), not the other way around. Educational objective: DNA synthesis can be arrested by replication inhibitors. Cells lacking the genes necessary to promote replication stability are more sensitive (damaged or affected) to the replication blockade induced by these agents, and recovery of replication may be impaired.

Passage the Bloom Syndrome Helicase (BLM) Transcript Is Catalytically Inactive in in Bloom