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.
-During HU treatment of PSNF5 cells, replication stress response factors would act at DNA fibers that resemble which rows in Figure 1B?

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.
-During HU treatment of PSNF5 cells, replication stress response factors would act at DNA fibers that resemble which rows in Figure 1B?

Quizplus · Accepted Answer

11ecba2d_0f4b_c0bf_a3bf_833daf7249c4_MD0008_00 During replication stress (HU treatment), DNA synthesis is arrested at the S phase of the eukaryotic cell cycle, and replication stress response factors are recruited to repair stalled forks. PSNF5 cells have normal BLM function, and BLM (required to repair DNA synthesis during replication stress) localizes to genomic regions of replication arrest. The DNA fibers depicted in Rows 5 and 6 show that only IdU incorporation occurred in the first 15 minutes and that CldU failed to integrate into the DNA strand during the post-replication stress period (85 minutes). Rows 5 and 6 therefore represent arrested replication forks during HU treatment, and response factors that repair replication stress (eg, BLM) act at these sites to restart DNA replication in S phase. (Choices A and B) Replication stress response factors would be active only at stalled forks, but these fibers show recovered replication in S phase. Rows 1 and 2 depict incorporation of IdU and CldU, signifying that the forks were able to restart after replication stress and integrate CldU into the DNA strand during replication. (Choice C) Rows 3 and 4 show only CldU incorporation, which means that new sites of replication arose after HU treatment stopped. In this case, stalled forks likely collapsed, eliminating the need for replication stress response factors. Educational objective: DNA replication (synthesis) occurs in S phase of the eukaryotic cell cycle.

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