Volume 18, Issue 3, Pages 285-292 (March 2010) A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader  Ah Young Park, Slobodan Jergic, Argyris Politis, Brandon T. Ruotolo, Daniel Hirshberg, Linda L. Jessop, Jennifer L. Beck, Daniel Barsky, Mike O'Donnell, Nicholas E. Dixon, Carol V. Robinson  Structure  Volume 18, Issue 3, Pages 285-292 (March 2010) DOI: 10.1016/j.str.2010.01.009 Copyright © 2010 Elsevier Ltd Terms and Conditions

Structure 2010 18, 285-292DOI: (10.1016/j.str.2010.01.009) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 Mass Spectra of the τ and γ Subunits in Isolation (A) Mass spectrum of τ in 0.1 M NH4OAc at pH 7.6 and 1 mM dithiothreitol. The major charge state series corresponds to a tetrameric form of τ. Series of τ in monomeric, dimeric, and trimeric forms are also observed as the minor charge state series. (B) Mass spectrum of γ in 0.1 M NH4OAc at pH 6.9. Well resolved charge states indicate that γ forms a well defined tetramer. (C) Mass spectrum of all four oligomeric species of γ in 1 M NH4OAc at pH 6.9. See also Table S1. Structure 2010 18, 285-292DOI: (10.1016/j.str.2010.01.009) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Mass Spectra of τ in the Presence of Various Subunits (A) In the presence of δ′, τ forms three subcomplexes, τ3δ′, τ2δ′, and τδ′ but not τ4δ′. (B) None of the oligomeric species of τ associates with δ. (C) The ψχ complex binds to τ, forming τ4ψχ and τ3ψχ. (D) δ does not associate with τ in the presence of ψχ. See also Figure S1 for the γ subunit interaction and Table S1. Structure 2010 18, 285-292DOI: (10.1016/j.str.2010.01.009) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 Assembly Pathway of the Clamp Loader and Mass Spectra of Complexes Formed Along the Pathway First, δ′ initiates the assembly by dissociating tetramers of τ into trimers, the functional unit of the clamp loader. Both δ or ψχ interact with the initial τ3δ′ subcomplex, resulting in either τ3δδ′ or τ3δ′ψχ subcomplexes. Further addition of ψχ or δ leads to the final clamp loader, τ3δδ′ψχ. Mass spectra are recorded for τ3δδ′ (A), τ3δ′ψχ (B), and τ3δδ′ψχ (C and D), when ψχ was added to (A) and δ to (B). For γ, analogous results were observed in Figure S2, suggesting identical assembly pathways. See also Table S1. Note that although all four species of oligomers of τ are present in solution, only tetramers of τ are shown initially. Structure 2010 18, 285-292DOI: (10.1016/j.str.2010.01.009) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 4 Subunit Exchange Dynamics between τ and γ (A) τ and γ undergo rapid subunit exchange at 0°C and form all five possible tetrameric species, τ4, τ3γ, τ2γ2, τγ3, and γ4, with the dead time of the experiment <15 s. See also Figure S3. (B) Mass spectra of τ3δδ′ in the presence of γ4. At t = 0, free δδ′ interacts with γ4 directly and forms the γ complex (bottom spectrum). At t = 1 hr, low intensity peaks are observed (not labeled, middle spectrum), which increase in intensity after 24 hr and are assigned to all four possible complexes, γ3δδ′, γτ2δδ′, γ2τδδ′, and τ3δδ′ (top spectrum). See also Table S1. Structure 2010 18, 285-292DOI: (10.1016/j.str.2010.01.009) Copyright © 2010 Elsevier Ltd Terms and Conditions