TheBiophysicalSociety of Japan General IncorporatedAssociation isomer of4-(phenylazo)-phenol <4-PAP) generated by UV irradiation inhibited ATPase activjty of Kifl 8A moTe effeetively than trans isomer of4-PAP Other azobenzen derivatives were also examined. Rad54 is a member of the SnMISwi2 family of chromatin remode[ing proteins, The members of this family belong to the superfamily2 he]iease, Nevertheless, they do not show helicase activity, Instead they translocate on DNA. Rad54 plays various

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... oles during repair of DNA double strand breaks with Rad51. In the initial phase of recombination, Rad54 binds to Rad51-ssDNA comp]ex, stabilizes the nucleoprotein filarnent and stirnulates the homejogy search by RadS 1-ssDNA. Interestingly, in the late phase of recombination, Rad54 disrupts the Rad51 nucleoprotein filament formed on dsDNA, which is inhibitory in the fo11owing steps. Alteration of protein-DNA interaction is one of the important roles of Rad54, In eukaryotes, the access to the target DNA is timited since the DNA is wrapped around a histone octamer. Therefore the DNA has to be exposed to a]low the DNA repair machinery to gain the access to the DNA. It has been reported that RadS4 has the abillty to slide nucleosomes along DNA. Hewever, the molecular mechanism is not yet understood. Here, we report that Rad54 generates 6-8 pN force and translecates along DNA by traeking the DNA helix. Our observations suggest that Rad54 conforms to the twist diffusion model when it slides nucleosomes, ln this model, when Rad54 encounters a nucleosome, RadS4 pulLs and twists the nucleosomal DNA. The twist defect generated by the action of Rad54 propagates along the histone surface, disrupting the DNA-histone contacts and re-establishing new contacts. As a Tesu]t, the nucleosome's position is changed. 1PSe46 Yusuke Moriguch {'sch. Addth. UC Davis, Uhiv.)RecBCD, a heterotrimeric protein complex of the E. coli, is a highly proccssive DNA helicase and nuclease that is involved in the seemingly conflicting functions of recembinational DNA repair and the degradation of DNA. These bio]ogical activities are regutated by a recombination-activating Chi-sequence coded on DNA, in which RecBCD digests away DNA until the Chi-sequence is recognized. At the Chi-sequence, RecBCD pauses, reduces the nuclease activity, and then switches its polarity to initiate the recombination process. Although these properties have been extensively studied by biochernical and biophysical methods, little is known about the detailed mechanisrn at the nanometer level. In this study, we applied high-speed atomic force microscopy {HS-AFM) to directly observe stmctura] dynamics of RecBCD at work. Last year, we reported successfu1 direct visualization ofDNA degradation processes by RecBCD, from which we obtained kinetjc parameters ofthe ATP hydrolysis similar to those reported previously. However, the detail strueture of RecBCD was not seen in the AFM movies due to the positional fluctuations of the molecules. This time, we developed a new experimenta1 setup in which Ni-NTA-containing lipid bilayers were used as a substrate on which ihe His-tagged RecBCD was specifically imrnobilized. This setup enabled us to distinguish the domain structures of RecBCD, In the presentation, we wM shew the obtained results together with the HS-AFM movies.