Bioinformatics and experimental workflow used for analysis of the coevolution of PCNA-partner interactions. Changes in the IDCL region of PCNA are analyzed by sequence alignment and PCA (step 1). Next, different chimeric PCNA variants are generated by substitution of the native IDCL region with IDCLs from different species (step 2). Such variants are characterized for PCNA-partner interactions and for in vivo function in S. cerevisiae as a model organism (step 3). Bar indicates a size of 5 microns.

In vivo phenotypic analysis of the chimeric S. cerevisiae PCNA variants from group I and group II species. (A) Sensitivity of yeast strains containing chimeric PCNA variants as the sole source of PCNA to DNA-damaging agents, including HU (Center) and MMS (Right). (B) Nonviable strains containing chimeric ScPCNA with the N. crassa or A. nidulans IDCL sequence. Yeast strains containing ScPCNA expressed from a URA3 plasmid and chimeric Scpcna genes expressed from a LEU2 plasmid are viable (Center). On selection for loss of WT PCNA using 5-FOA, chimeric PCNA strains are deemed nonviable (Right).

Representative yeast two-hybrid analysis of chimeric and WT PCNA variants interacting with group I and group II PCNA partners. (A) Representative analysis of the interaction of chimeric PCNA with S. cerevisiae RAD27 (Left) or POL32 (Right). A full-yeast two-hybrid analysis is provided in Table 1. (B) Representative analysis of the interactions of ScPCNA or SpPCNA with S. cerevisiae Rrm3 and Msh6. SpPCNA exhibits higher interaction levels with the two partners, relative to ScPCNA (results of a full-yeast two-hybrid analysis are provided in Table S1). (C) Yeast two-hybrid analysis of the SpPCNA, ScPCNA (POL30), and chimeric SpPCNA variants interacting with CDC9, RAD27, POL32, and UNG1 orthologs in S. pombe. Interactions can be detected in most cases only with SpPCNA but not with ScPCNA or the chimeric SpPCNA variants containing the ScPCNA IDCL region (pcn1-POL30-IDCL; Table 2).

Bioinformatics and experimental workflow used for analysis of the coevolution of PCNA-partner interactions Changes in the sequences of the IDCL region of the PCNA model protein network are analyzed (step 1). Next, different chimeric PCNA variants are generated by substitution of the native IDCL region with IDCL from different species (step 2). Such variants are characterized for PCNA-partner interactions and for in vivo function in S. cerevisiae as a model organism (step 3).

Bioinformatics analysis of PCNA IDCL sequences across different species indicates divergence into two groups. (A) Sequence alignment of the IDCL region indicates changes mainly in four amino acids (highlighted in color) that can be used to separate the sequences into two groups (group I and group II; highlighted in red and blue, respectively). (B) Species phylogenetic tree containing 23 well-annotated fungal species (11). Group I and group II are highlighted in red and blue, respectively. (C) PCA of PCNA IDCL sequences from 23 species, including 15 unique sequences, indicates divergence into two groups; representatives of group I and group II are highlighted. The ellipse was generated by measuring 3 SDs around the centroid of each group, using Partek. (D) Similar analysis performed on IDCL sequences from 53 species, including 21 unique sequences (Fig. S1) found on BLAST analysis of fungal sequences (http://www.yeastgenome.org/). PCA was performed using the Jalview and Partek programs. The color scheme of amino acids in A is according to Lesk (28). Small nonpolar residues (G, A, S, T) are highlighted in yellow, hydrophobic residues (C, V, I, L, P, F, Y, M, W) are highlighted in green, polar residues (N, Q, H) are highlighted in magenta, negatively charged residues (D, E) are highlighted in red, and positively charged residues (K, R) are highlighted in blue.

In vivo phenotypic analysis of WT PCNAs from group I and group II species. (A) HU (Center) and MMS (Right) sensitivity of yeast strains containing the ScPCNA, KwPCNA, AgPCNA, or DhPCNA variants as the sole source of PCNA. (B) Nonviable strains containing NcPCNA. (C) Nonviability of the S. cerevisiae strain containing YlPCNA is suppressed by the replacement of the native IDCL region with the ScPCNA (POL30) IDCL region (Ylpcna-POL30-IDCL), indicating the importance of this region in facilitating strain survival (the generation of S. cerevisiae strains containing PCNA variants as a sole source of PCNA in the cell was performed as described in Fig. 3 and in the main text). (D) HU (Center) and MMS (Right) sensitivity of strains containing the SpPCNA the chimeric Ylpcna-POL30-IDCL, or the chimeric pcn1-POL30-IDCL gene. The sensitivity of the SpPCNA-containing strain is suppressed by replacement of its native IDCL region with the POL30 IDCL region (pcn1-POL30-IDCL).

In vitro evolution of Y. lipolytica PCNA for complementation of ScPCNA in S. cerevisiae. (A) Sequence alignment of 9 unique mutants identified following selection for viability. The mutated positions are mostly hydrophobic and are highlighted in color (using the color scheme described in the legend for Fig. 2). (B) PCA analysis of the 9 unique sequences of the evolved clones, together with the 23 natural IDCL sequences (described in Fig. 2). The analysis indicates that the evolved Ylpcna mutants fall into a unique group that is different from the two natural groups, except for YlPCNA11, which is significantly different in its sequence. (C) HU, MMS, and 4-nitroquinoline-N-oxide (4NQO) sensitivity of the evolved Ylpcna mutants.