Journal: International Journal of Biological Sciences

Article Title: Budding Yeast Ubiquitin Ligases Rad5 and Rad18 Bind a Novel PCNA Surface, which Is Required for their Functions in DNA-Damage Tolerance

doi: 10.7150/ijbs.124441

Figure Lengend Snippet: Identification and characterization of an LxLF motif from Rad5 and Rad18 . ( A ) Sequence alignment of the mapped minimum PCNA-binding regions in Rad5 and Rad18. The sequence alignment was performed by BioEdit 7.2. Residues highlighted in black are identical sequences and those in grey are conserved residues. ( B , C ) Physical interactions between Rad5/Rad18 and Pol30 by a Y2H assay. The 3A mutation indicates LxLF-to-AxAA amino acid substitution. Rad5 and Rad18 interactions with Smt3 served as positive controls, respectively. ( D ) Effects of 3A and 3E amino acid substitutions on the physical interaction between Rad5-N431 and Pol30 by a Y2H assay. The 3E mutation contains R187E, R229E, R241E triple substitutions; 3AE contains both 3A and 3E mutations. ( E , F) Assess physical interactions between Rad5 (177-348)/Rad18-N160 and Pol30 by a GST affinity pulldown assay. Experimental conditions were as described in Figure B and D. For the Y2H assay, at least two sets of independent transformants were tested with comparable results, and only one set of representative images is presented.

Article Snippet: Multiple alignments were performed and presented by BioEdit 7.2 ( https://bioedit.software.informer.com/7.2/ ).

Techniques: Sequencing, Binding Assay, Y2H Assay, Mutagenesis