Abstract
Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif1 unwinds forked dsDNA remains elusive. Here we report the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA. Two interacting BaPif1 molecules are bound to each fork of the partially unwound dsDNA, and interact with the 5′ arm and 3′ ss/dsDNA respectively. Each of the two BaPif1 molecules is an active helicase and their interaction may regulate their helicase activities. The binding of BaPif1 to the 5′ arm causes a sharp bend in the 5′ ss/dsDNA junction, consequently breaking the first base-pair. BaPif1 bound to the 3′ ss/dsDNA junction impacts duplex unwinding by stabilizing the unpaired first base-pair and engaging the second base-pair poised for breaking. Our results provide an unprecedented insight into how two BaPif1 coordinate with each other to unwind the forked dsDNA.
Highlights
Pif[1] plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif[1] unwinds forked dsDNA remains elusive
The asymmetric unit of the crystal structure contains two BaPif[1] molecules plus one DNA strand, the model of the biologically meaningful Bacteroides sp Pif1 (BaPif1)–10dT-fdsDNA complex is generated by the application of a crystallographic twofold operation
The conformations of two BaPif[1] molecules are essentially identical and are similar to those observed in our previous structures of BaPif[1] in complex with ssDNA and ADP·AlF4– 25, suggesting that the BaPif[1] molecules in these structures undergo similar conformational change upon concomitant binding of ATP and DNA (Supplementary Fig. 1b)
Summary
Pif[1] plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif[1] unwinds forked dsDNA remains elusive. We report the structure of Bacteroides sp Pif[1] (BaPif1) in complex with a symmetrical double forked dsDNA. The binding of BaPif[1] to the 5′ arm causes a sharp bend in the 5′ ss/dsDNA junction, breaking the first base-pair. BaPif[1] bound to the 3′ ss/dsDNA junction impacts duplex unwinding by stabilizing the unpaired first base-pair and engaging the second base-pair poised for breaking. The signature motif of ScPif[1] is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity[23] while the signature motif of Pfh[1] is necessary for both protein displacement and helicase unwinding activities[24]
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