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Bustamante/AAAS3 b6 U. a( b, i7 }0 q# G* ]. r
2 m2 I g# D; i; `- XDNA compaction, say Ryan Case, Yun-Pei Chang, Nicholas Cozzarelli, Carlos Bustamante, and colleagues (University of California, Berkeley, CA), may work via a cooperative caterpillar-like mechanism.
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The caterpillar is formed by multiple copies of condensin protein—in this case MukBEF—with each V-shaped condensin contributing two legs. Compaction occurs when the caterpillar's legs snap together.
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"The main problem," says Bustamante, "is that we have not had any bulk assay for this protein. We decided to do a rather risky search for a single molecule assay."
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+ U, `' T0 O9 o8 N, K2 ^The assay involved binding MukBEF to DNA and then holding both ends of the compacted DNA strand using a dual beam optical trap. As the DNA strand was pulled, it gave way in a sawtooth pattern as individual copies of MukBEF stayed on the DNA but splayed apart to release their captured loops of DNA. The size of each peak depended on just how much DNA was released per event.4 C4 ^' }6 V6 x/ \) r
8 h5 D1 c% r3 a1 D/ K5 }- H! tMukBEF binding was cooperative, so the proteins opened up in a set sequence starting at one end. Thus, after recondensing, restretching gave the exact same sawtooth pattern.% A! x9 O8 r6 v3 {
* {8 S+ U- C) ]3 y4 T3 lThe open V of MukBEF may span 200bp or more. ATP binding (but not hydrolysis) may drive compaction by closing the V. In vivo, however, DNA is already compacted by other proteins and supercoiled, so each condensin would reach across many kilobases of DNA. Bustamante hopes to establish a bulk assay for condensin action so that he can see this process occur in real time.* s9 Y6 Z/ Z1 N v$ }1 a
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Reference:' h( b4 x! | t, w6 C, x; `" s
' ~( p3 T" F- C2 n8 pCase, R.B., et al. 2004. Science. 10.1126/science.1098225.(DNA compacted with MukBEF extends in an ) |
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发表于 2009-3-6 08:11
发表于 2015-5-23 17:27
