Processivity clamps such as proliferating cell nuclear antigen(PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1(9-1-1) act as versatile scaffolds in the recruitment of proteins in DNA replication, cell-cycle control and DNA repair. Association and handoff of DNA-editing enzymes e.g. flap-endonuclease-1(FEN1) with sliding clamps are key processes in PCNA biology. We have chosen an integrative computational and experimental approach to define the assemblies of FEN1 with DNA substrates and either PCNA or 9-1-1. Fully atomistic models of these complexes were developed and refined through extensive MD simulations and used in conjunction with single-particle electron microscopy(EM) data to generate an EM map of the 9-1-1/Fen1/DNA assembly at 18.5� resolution. Flexible fitting into the density allowed further improvement of the models. Comparative structural analysis revealed key differences in the orientation and interactions of FEN1 with the two clamps, which could explain observed differences in the stimulation of the enzyme activity by PCNA and 9-1-1.

Electron microscopy and computational modeling reveal key structural aspects of the ternary assemblies of flap endonuclease 1 with sliding clamps and DNA

243rd National Meeting (2012)

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