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Some helicases continually bang their heads against the wall, if a new report from Sua Myong, Taekjip Ha (University of Illinois, Urbana-Champaign, IL), and colleagues is any indication. The Rep helicase, the group finds, repeatedly motors along a track of DNA, hits an obstacle, and returns to square one.% m8 U) _) ~: x6 }
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Not all helicases can unwind DNA, but they can all motor along it. While studying how Rep motors in a 5' direction on single-stranded (ss) DNA, Ha's group noticed that a duplex DNA obstacle did not knock Rep off its template. Their FRET analysis instead suggested that the helicase returned to its original binding site and tried again.: n% q* `2 O. }" k! Q
3 Z- n4 i8 `$ _1 [1 v# XUnlike the 5' motoring, the return step was almost instantaneous. "At first it seemed like Rep was doing some sort of quantum tunneling," says Ha, who is a physicist by training. But more FRET studies cleared up the situation—Rep, it seemed, was transiently bound both to DNA near the obstacle and to its initial 3' binding site, creating a ssDNA loop.7 W5 O$ C3 A4 P5 g2 A
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Closing of a regulatory region of Rep called domain 2B coincided with the sudden 3' end capture. As Rep approached the obstacle, 2B was increasingly likely to be in a closed conformation. Ha guesses that collisions with the duplex—which would be more frequent as Rep gets closer—might push 2B into its closed position. This closed conformation might then trigger high-affinity binding to 3' ends of ssDNA—a form of DNA that is, says Ha, "very flexible, like spaghetti.") R% f* j& p( x$ f" i
: q# ]5 l6 {) {5 ?' D% M+ Y% xIn addition to free 3' ends, Rep also has a high affinity for the three-way junctions at stalled replication forks. The group found that Rep shuttled repeatedly between a fork structure and an Okazaki fragment (the equivalent of the duplex obstacle).
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. t! K! o' F# p9 L" a2 Y) f% G4 EThe ssDNA at stalled replication forks is a target for RecA binding, which promotes recombination. But Rep prevented RecA filament formation. The findings might thus explain why Rep mutations lead to increased recombination in bacteria. If so, perhaps the clearing, not the unwinding, of DNA is this helicase's main duty.(Collisions with duplex DNA close Rep's 2) |
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发表于 2009-3-6 08:56
发表于 2015-6-25 19:43
