A structural phylogenetic tree of Rad52 and its annealase superfamily

Abstract

Rad52, a highly conserved eukaryotic protein, plays a crucial role in DNA repair, particularly in double-strand break repair. Recent findings reveal that its distinct structural features, including a characteristic β-sheet and β-hairpin motif, are shared with the lambda phage single-strand annealing protein, Redβ, and other prokaryotic single-strand annealing proteins (SSAPs), indicating a common superfamily. Our analysis of over 10,000 SSAPs across all domains of life supports this hypothesis, confirming the presence of the characteristic motif despite variations in size and composition. We found that archaea, representing only 1% of the studied proteins, exhibit most of these variations as reflected by their spread across the phylogenetic tree, whereas eukaryotes exhibit only Rad52. By examining four representative archaeal SSAPs, we elucidate the structural relationship between eukaryotic and bacterial SSAPs, highlighting differences in β-sheet size and β-hairpin complexity. Furthermore, we identify an archaeal SSAP with a predicted structure nearly identical to human Rad52. Together with a screen of over 100 million unannotated proteins for potential SSAP candidates, our computational analysis complements the existing sequence and structural evidence supporting orthology among five SSAP families: Rad52, Redβ, RecT, Erf, and Sak3.

Keywords: AlphaFold; DNA repair; Homology; Rad52; SSAP.

Graphical abstract

Fig. 1

(A) 10,280 SSAPs clustered by structural similarity confirm the definition of SSAP families: RecT (brown), Redβ (red), Erf (green), Sak3 (purple), and Rad52 (blue). RecT/Redβ are clearly separated from Erf, Sak3, and Rad52. Sak3 is placed within the Rad52 cluster. Eukaryotic Rad52 (light blue) and prokaryotic Rad52 (dark blue) are resolved together, while the RDM1 subgroup (turquoise), which has an additional RNA-binding motif, is a sister clade. The 124 archaeal SSAPs are labeled by their IDs, showing that they cover the full diversity of the 10,280 SSAPs. (B) Close-up of the 124 archaeal SSAPs clustered by structural similarity, with the four selected representatives labeled by their identifiers. (C) The four selected representatives with their β-hairpin and β-sheet motifs, serving as references in subsequent analyses.

Fig. 2

Scatter plot of structural similarity (TM-score) for each of the 10,280 SSAPs compared to the RecT (brown) and Redβ (red) representatives. The RecT and Redβ SSAPs are clearly distinct from other families and are also separated from each other. On the right, the three plots show the same settings as on the left, but for each superkingdom separately.

Fig. 3

Scatter plot of structural similarity (TM-score) for each of the 10,280 SSAPs compared to the long and short β-sheet Rad52 representatives. Most SSAPs exhibit significant structural similarity. Bacterial Rad52 SSAPs (dark blue) are similar to both the long and short β-sheet Rad52 representatives. Eukaryotic Rad52 SSAPs (medium blue) are also highly similar to both but display greater similarity to the long β-sheet Rad52. Due to its additional RNA-binding domain, the Rad52 subfamily RDM1 (light blue) is more dissimilar to both representatives. On the right, the three plots show the same settings as on the left but are separated by superkingdom.

Fig. 4

Distribution of alignment lengths (number of residues aligned) against four representative SSAPs. Structural alignment lengths are shown for: (A) RecT, (B) Redβ, (C) Rad52 (long β-sheet), and (D) Rad52 (short β-sheet).

Fig. 5

Scatter plot of structural similarity (TM-score) for all AlphaFold structures, including 117.5 million unreviewed proteins, compared to the two representative Rad52 SSAPs. The bulk of the proteins have TM-scores below 0.5. Many known SSAPs have TM-scores above 0.5. Among the top-scoring proteins, 2,164 (1,458) proteins have a TM-score greater than 0.7 against the long (short) β-sheet Rad52 representative. These are potential novel SSAP candidates.

Fig. 6

(A) Structural alignment of the archaeal SSAP A0A2D6XHC3 (green) with human Rad52 (blue) shown from different projection angles. The two structures align closely, with a TM-score of 0.82. (B) Confidence score (pLDDT) of the AlphaFold prediction. Red indicates very high confidence, while blue indicates low confidence. The discussed motif is highlighted in red, indicating a high-confidence prediction.