Figure 7.

Phosphorylation at a single position in RPA70 alters access to the ssDNA with minimal alternations of end-to-end distance. (A) SDS-PAGE and Phos-tag SDS-PAGE analysis of RPA and RPA-pSer³⁸⁴ proteins show selective shift of the RPA70 band only in the RPA-pSer³⁸⁴ sample confirming 100% incorporation of pSer. (B) Western blotting of RPA and RPA-pSer³⁸⁴ with an antibody specific to pSer³⁸⁴ confirms the site-specific phosphorylation. (C, D) Cross-linking mass spectrometry (XL-MS) analysis of RPA in the absence or presence of ssDNA (dT)₂₅. A direct comparison of the XLs between RPA and the RPA-(dT)₂₅ experiments are presented with XLs unique to each condition denoted in blue. (F) A similar comparative XL-MS analysis RPA-pSer³⁸⁴ and the RPA-pSer³⁸⁴-(dT)₂₅ complex is shown and the XLs unique to each condition is shown in blue. The XL patterns show differences in ssDNA driven changes upon phosphorylation.