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 the 85-minute period, deoxyribonucleoside triphosphates (dNTPs) are added and DNA synthesis is allowed to continue for 20 minutes. Which of the following processes is most likely to occur during the new replication period? The structure of CldU is shown 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 the 85-minute period, deoxyribonucleoside triphosphates (dNTPs) are added and DNA synthesis is allowed to continue for 20 minutes. Which of the following processes is most likely to occur during the new replication period? The structure of CldU is shown below.

Quizplus · Accepted Answer

11ecba2d_0f4c_ab21_a3bf_63538a9e05a7_MD0008_00 During replication, DNA polymerase attaches uncoupled deoxyribonucleoside triphosphates (dNTPs) to the growing DNA strand. Each dNTP (dTTP, dATP, dCTP, and dGTP) is composed of a base, a deoxyribose sugar, and three phosphate (PO₄) groups. CldU is a thymidine analog, meaning that it can be incorporated into the growing DNA strand in place of thymidine, which bonds to adenosine via two hydrogen bonds. After 85 minutes, the time-controlled incorporation of CldU into the growing DNA strand stops. Therefore, thymidine would be incorporated into the growing strand instead of CldU during the new 20-minute replication period. Based on the AT/CG rule of complementary base pairing, a free dNTP enters the catalytic site of DNA polymerase and forms hydrogen bonds with a complementary nucleotide on the parent (template) DNA strand. The 3′ OH from the last nucleotide of the new growing strand "attacks" the 5′ PO₄ group of the incoming dNTP (Choice D). As a result, two phosphate groups are cleaved off the dNTP, resulting in a nucleoside monophosphate molecule and in the release of water, pyrophosphate (PP_i), and energy (exergonic process). PP_i is further cleaved into two phosphate groups to release more energy. The energy generated from these reactions is used to form a covalent phosphodiester bond between the last nucleotide of the growing strand and the incoming dNTP (endergonic process). Once the phosphodiester bond is formed, the enzyme proceeds to couple the next free dNTP to the new growing strand. (Choices A and B) dNTP cleavage is exergonic, but phosphodiester bond formation is endergonic. CldU is no longer incorporated into the growing DNA strand after 85 minutes; therefore, it is not involved in the reactions necessary for nucleotide coupling during DNA synthesis in the new 20-minute replication period. Educational objective: During replication, DNA polymerase joins uncoupled deoxyribonucleoside triphosphates to the new DNA strand. As a result, the exergonic release of a pyrophosphate molecule leads to the formation of a covalent phosphodiester bond between the last nucleotide of the growing strand and the incoming nucleotide.

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