title: Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1.
abstract: SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.
authors.affiliation: Department of Biochemistry, ‡Department of Biological Sciences, §Department of Chemistry, and ∥Center for Structural Biology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Feldkamp
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Mason
authors.forename: Brandt F
authors.initials: BF
authors.lastname: Eichman
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D051716:Replication Protein A / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D051716:Replication Protein A; C412482:SMARCAL1 protein, human; C516794:RPA2 protein, human; D004265:DNA Helicases
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