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binding of the Mcm4/6/7 double hexamer to bubble suggests that each hexamer catalyzes unwinding of one of the two duplex segments. We asked whether activation of both hexamers is required for unwinding of the bubble substrate. The preferred bubble substrate which is efficiently unwound by Mcm4/6/7 contains thymine stretches on both strands of the single-stranded segment. We constructed a hetero-bubble (Bub66/T–G-rich) in which one strand is T-rich (same as Bub66/T-rich) and the other strand is G-rich (same as Bub66/G-rich) (13), and helicase assays were conducted with increasing amount of Mcm4/6/7. Only weak unwinding was detected on this substrate compared to Bub66/T-rich (Figure 3A). Since Mcm4/6/7 can bind to Bub66/T–G-rich and forms a double hexameric complex (data not shown), the defective unwinding is not due to inefficient loading of the helicase onto the substrate. If one hexamer interacting with the thymine-rich strand dissociates from the other hexamer and displaces only one of the duplex segments, then it would result in generation of Y-fork structures which could not be further displaced due to the presence of G-rich 3′-tail and would appear on the gel. However, we did not detect such intermediates during the course of the experiment (Figure 3B), suggesting that bubble can be displaced efficiently only when two molecules of the Mcm4/6/7 hexamers are simultaneously activated."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T4698","span":{"begin":10,"end":17},"obj":"CHEMINF_GO_EXT:chemical_binding_or_bond_formation"},{"id":"T4699","span":{"begin":25,"end":29},"obj":"PR_EXT:000010246"},{"id":"T4700","span":{"begin":25,"end":33},"obj":"GO:0097373"},{"id":"T4701","span":{"begin":34,"end":48},"obj":"GO_EXT:double_hexameric_macromolecular_complex"},{"id":"T4702","span":{"begin":78,"end":85},"obj":"GO_EXT:hexameric_macromolecular_complex"},{"id":"T4703","span":{"begin":86,"end":95},"obj":"GO_MOP_EXT:catalysis"},{"id":"T4704","span":{"begin":124,"end":130},"obj":"SO:0000985"},{"id":"T4705","span":{"begin":177,"end":185},"obj":"GO_EXT:hexameric_macromolecular_complex"},{"id":"T4706","span":{"begin":300,"end":304},"obj":"PR_EXT:000010246"},{"id":"T4707","span":{"begin":300,"end":308},"obj":"GO:0097373"},{"id":"T4708","span":{"begin":318,"end":325},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4709","span":{"begin":359,"end":374},"obj":"SO:0000984"},{"id":"T4710","span":{"begin":422,"end":423},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4711","span":{"begin":424,"end":425},"obj":"CHEBI_EXT:guanine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4712","span":{"begin":455,"end":456},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4713","span":{"begin":477,"end":478},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4714","span":{"begin":509,"end":510},"obj":"CHEBI_EXT:guanine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4715","span":{"begin":531,"end":532},"obj":"CHEBI_EXT:guanine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4716","span":{"begin":549,"end":557},"obj":"GO_EXT:0004386"},{"id":"T4717","span":{"begin":606,"end":610},"obj":"PR_EXT:000010246"},{"id":"T4718","span":{"begin":606,"end":614},"obj":"GO:0097373"},{"id":"T4719","span":{"begin":685,"end":686},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4720","span":{"begin":711,"end":715},"obj":"PR_EXT:000010246"},{"id":"T4721","span":{"begin":711,"end":719},"obj":"GO:0097373"},{"id":"T4722","span":{"begin":724,"end":728},"obj":"CHEMINF_GO_EXT:chemical_binding_or_bond_formation"},{"id":"T4723","span":{"begin":738,"end":739},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4724","span":{"begin":740,"end":741},"obj":"CHEBI_EXT:guanine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4725","span":{"begin":759,"end":783},"obj":"GO_EXT:double_hexameric_macromolecular_complex"},{"id":"T4726","span":{"begin":867,"end":875},"obj":"GO_EXT:0004386"},{"id":"T4727","span":{"begin":903,"end":910},"obj":"GO_EXT:hexameric_macromolecular_complex"},{"id":"T4728","span":{"begin":932,"end":939},"obj":"CHEBI_EXT:thymine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4729","span":{"begin":979,"end":986},"obj":"GO_EXT:hexameric_macromolecular_complex"},{"id":"T4730","span":{"begin":1017,"end":1023},"obj":"SO:0000985"},{"id":"T4731","span":{"begin":1074,"end":1078},"obj":"GO:0005657"},{"id":"T4732","span":{"begin":1150,"end":1151},"obj":"CHEBI_EXT:guanine_nucleobase_or_nucleoside_or_nucleotide_molecular_entity_or_group"},{"id":"T4733","span":{"begin":1355,"end":1364},"obj":"CHEBI:36357"},{"id":"T4734","span":{"begin":1372,"end":1376},"obj":"PR_EXT:000010246"},{"id":"T4735","span":{"begin":1372,"end":1380},"obj":"GO:0097373"},{"id":"T4736","span":{"begin":1381,"end":1389},"obj":"GO_EXT:hexameric_macromolecular_complex"}],"text":"Symmetric binding of the Mcm4/6/7 double hexamer to bubble suggests that each hexamer catalyzes unwinding of one of the two duplex segments. We asked whether activation of both hexamers is required for unwinding of the bubble substrate. The preferred bubble substrate which is efficiently unwound by Mcm4/6/7 contains thymine stretches on both strands of the single-stranded segment. We constructed a hetero-bubble (Bub66/T–G-rich) in which one strand is T-rich (same as Bub66/T-rich) and the other strand is G-rich (same as Bub66/G-rich) (13), and helicase assays were conducted with increasing amount of Mcm4/6/7. Only weak unwinding was detected on this substrate compared to Bub66/T-rich (Figure 3A). Since Mcm4/6/7 can bind to Bub66/T–G-rich and forms a double hexameric complex (data not shown), the defective unwinding is not due to inefficient loading of the helicase onto the substrate. If one hexamer interacting with the thymine-rich strand dissociates from the other hexamer and displaces only one of the duplex segments, then it would result in generation of Y-fork structures which could not be further displaced due to the presence of G-rich 3′-tail and would appear on the gel. However, we did not detect such intermediates during the course of the experiment (Figure 3B), suggesting that bubble can be displaced efficiently only when two molecules of the Mcm4/6/7 hexamers are simultaneously activated."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T4676","span":{"begin":25,"end":29},"obj":"PR:000010246"},{"id":"T4677","span":{"begin":25,"end":33},"obj":"GO:0097373"},{"id":"T4678","span":{"begin":124,"end":130},"obj":"SO:0000985"},{"id":"T4679","span":{"begin":300,"end":304},"obj":"PR:000010246"},{"id":"T4680","span":{"begin":300,"end":308},"obj":"GO:0097373"},{"id":"T4681","span":{"begin":359,"end":374},"obj":"SO:0000984"},{"id":"T4682","span":{"begin":606,"end":610},"obj":"PR:000010246"},{"id":"T4683","span":{"begin":606,"end":614},"obj":"GO:0097373"},{"id":"T4684","span":{"begin":711,"end":715},"obj":"PR:000010246"},{"id":"T4685","span":{"begin":711,"end":719},"obj":"GO:0097373"},{"id":"T4686","span":{"begin":1017,"end":1023},"obj":"SO:0000985"},{"id":"T4687","span":{"begin":1074,"end":1078},"obj":"GO:0005657"},{"id":"T4688","span":{"begin":1355,"end":1364},"obj":"CHEBI:36357"},{"id":"T4689","span":{"begin":1372,"end":1376},"obj":"PR:000010246"},{"id":"T4690","span":{"begin":1372,"end":1380},"obj":"GO:0097373"}],"text":"Symmetric binding of the Mcm4/6/7 double hexamer to bubble suggests that each hexamer catalyzes unwinding of one of the two duplex segments. We asked whether activation of both hexamers is required for unwinding of the bubble substrate. The preferred bubble substrate which is efficiently unwound by Mcm4/6/7 contains thymine stretches on both strands of the single-stranded segment. We constructed a hetero-bubble (Bub66/T–G-rich) in which one strand is T-rich (same as Bub66/T-rich) and the other strand is G-rich (same as Bub66/G-rich) (13), and helicase assays were conducted with increasing amount of Mcm4/6/7. Only weak unwinding was detected on this substrate compared to Bub66/T-rich (Figure 3A). Since Mcm4/6/7 can bind to Bub66/T–G-rich and forms a double hexameric complex (data not shown), the defective unwinding is not due to inefficient loading of the helicase onto the substrate. If one hexamer interacting with the thymine-rich strand dissociates from the other hexamer and displaces only one of the duplex segments, then it would result in generation of Y-fork structures which could not be further displaced due to the presence of G-rich 3′-tail and would appear on the gel. However, we did not detect such intermediates during the course of the experiment (Figure 3B), suggesting that bubble can be displaced efficiently only when two molecules of the Mcm4/6/7 hexamers are simultaneously activated."}

{"project":"2_test","denotations":[{"id":"15917436-14609960-76775513","span":{"begin":540,"end":542},"obj":"14609960"}],"text":"Symmetric binding of the Mcm4/6/7 double hexamer to bubble suggests that each hexamer catalyzes unwinding of one of the two duplex segments. We asked whether activation of both hexamers is required for unwinding of the bubble substrate. The preferred bubble substrate which is efficiently unwound by Mcm4/6/7 contains thymine stretches on both strands of the single-stranded segment. We constructed a hetero-bubble (Bub66/T–G-rich) in which one strand is T-rich (same as Bub66/T-rich) and the other strand is G-rich (same as Bub66/G-rich) (13), and helicase assays were conducted with increasing amount of Mcm4/6/7. Only weak unwinding was detected on this substrate compared to Bub66/T-rich (Figure 3A). Since Mcm4/6/7 can bind to Bub66/T–G-rich and forms a double hexameric complex (data not shown), the defective unwinding is not due to inefficient loading of the helicase onto the substrate. If one hexamer interacting with the thymine-rich strand dissociates from the other hexamer and displaces only one of the duplex segments, then it would result in generation of Y-fork structures which could not be further displaced due to the presence of G-rich 3′-tail and would appear on the gel. However, we did not detect such intermediates during the course of the experiment (Figure 3B), suggesting that bubble can be displaced efficiently only when two molecules of the Mcm4/6/7 hexamers are simultaneously activated."}