Remote Homology Detection Identifies a Eukaryotic RPA DBD-C-like DNA Binding Domain as a Conserved Feature of Archaeal Rpa1-Like Proteins

Abstract

The eukaryotic single-stranded DNA binding factor replication protein A (RPA) is essential for DNA replication, repair and recombination. RPA is a heterotrimer containing six related OB folds and a winged helix-turn-helix (wH) domain. The OB folds are designated DBD-A through DBD-F, with DBD-A through DBD-D being directly involved in ssDNA binding. DBD-C is located at the C-terminus of the RPA1 protein and has a distinctive structure that includes an integral C4 zinc finger, while the wH domain is found at the C-terminus of the RPA2 protein. Previously characterised archaeal RPA proteins fall into a number of classes with varying numbers of OB folds, but one widespread class includes proteins that contain a C4 or C3H zinc finger followed by a 100-120 amino acid C-terminal region reported to lack detectable sequence or structural similarity. Here, the sequences spanning this zinc finger and including the C-terminal region are shown to comprise a previously unrecognised DBD-C-like OB fold, confirming the evolutionary relatedness of this group of archaeal RPA proteins to eukaryotic RPA1. The evolutionary relationship between eukaryotic and archaeal RPA is further underscored by the presence of RPA2-like proteins comprising an OB fold and C-terminal winged helix (wH) domain in multiple species and crucially, suggests that several biochemically characterised archaeal RPA proteins previously thought to exist as monomers are likely to be RPA1-RPA2 heterodimers.

Keywords: DNA repair; DNA replication; OB fold; RPA; archaea; ssDNA; winged helix domain.

FIGURE 1

DBD-C-like OB fold in the archaeal RPA1-like proteins (A) Domain structure of eukaryotic and archaeal RPA proteins: S. cerevisiae RPA complex components RPA1, RPA2, and RPA3 (boxed with OB folds labelled DBD-A through-F) and representative archaeal zinc finger-containing RPA proteins from Methanocaldococcus jannaschii (MjaRPA), Methanosarcina acetivorans (MacRPA2), and Haloferax volcanii (HvoRpa3). The location of zinc finger (ZnF) and RPA2 winged helix (wH) domains are indicated (B) Crystal structure of the trimerization core of Ustilago maydis RPA bound to single-stranded DNA (PDB 4GNX) with OB folds from RPA1 (DBD-A, DBD-B, and DBD-C), RPA2 (DBD-D), and RPA3 (DBD-E) shown in different colours (Fan and Pavletich, 2012). The RPA1 zinc finger motif is shown in orange and single-stranded DNA in yellow (C) Protein sequence alignment of the C-terminal domains of archaeal and eukaryotic RPA proteins. UniProtKB or PDB accession numbers are shown on the left, followed by the organism and protein name. The positions of the first and last residues of the aligned region of the corresponding protein are indicated, with the number in brackets indicating the full length of the protein chain. Hydrophobic amino acids (AILVFMWYCH) are highlighted in yellow and smaller amino acids (GVACDENPST) highlighted in green when present in 9 of the 12 sequences shown. Residues (Y479, Y487, I524, W537, F541, F590) identified as being involved in ssDNA binding by the U. maydis RPA1 protein are indicated by red dots. The secondary structures of human, S. cerevisiae and U. maydis proteins are indicated (H = α-helix; E = ß-strand) and were taken from the indicated PDB entries. Abbreviations: Mja, M. jannaschii; Mth, Methanothermobacter thermautotrophicus; Mka, Methanopyrus kandleri; Mac, M. acetivorans; Hvo, H. volcanii; Pfu, Pyrococcus furiosus; Fac, Ferroplasma acidarmanus; Hs, human; Sc, Saccharomyces cerevisiae; and Um, Ustilago maydis.

FIGURE 2

Modelled structure of Mja RPA DBD-C fold (A) Structure of U. maydis Rpa1 DBD-C fold bound to 16 nucleotides of ssDNA (shown as grey mesh, for clarity only bound nucleotides 10–25 from PDB: 4GNX are shown). Sidechains of amino acid residues implicated in DNA binding by base stacking are shown in stick form and labelled (see also Figure 1C). The zinc ion is shown is magenta (B) Modelled structure of M. jannaschii RPA DBD-C fold. The model was generated using HHpred (Zimmermann et al., 2017) and MODELLER 10.1 (Webb and Sali, 2021). Residues corresponding to the DNA binding residues in the U. maydis Rpa1 DBD-C are highlighted. (C) Conservation of ssDNA- and zinc-binding amino acids between Um Rpa1 and Mja RPA. The bases stacked by the amino acids listed are shown to the right.

FIGURE 3

Winged helix-turn-helix (wH) domain in the archaeal RPA2-like proteins (A) Chromosomal organisation of ORFs encoding biochemically characterised archaeal RPA1-like and RPA2-like proteins (shown in green and blue, respectively). Linked (adjacent) and unlinked ORFs are shown (B) Right: Crystal structure of the wH domain at the C-terminus of the human RPA32 protein (PDB: 4OU0, RPA32 is the human orthologue of yeast RPA2). The structure comprises a three-helix bundle (α1–α3) capped by a ß-hairpin (β1–β3) (Feldkamp et al., 2014). Left: Structural model of the C-terminal wH domain (amino acids 169–225) of the M. jannaschii RPA2-like protein MJ_1654 based on its similarity to human RPA32 (amino acids 206–270, PDB: 4OU0). The model was generated using HHpred (Zimmermann et al., 2017) and MODELLER 10.1 (Webb and Sali, 2021) (C) Protein sequence alignment (as Figure 1, but with colouring applied when similar amino acids are present in seven of nine RPA2-like RPA proteins shown) of the putative wH domains of archaeal and eukaryotic RPA proteins, along with the human RPA2-like protein STN1 (Lim et al., 2020) and the M. thermautotrophicus MCM wH domains (Wiedemann et al., 2015). Alignments reflect the output of HHpred searches (Table 1) except for the Mac MA_318 and Hvo Rpap3 proteins which were manually aligned. The full ID of the Ferroplasma acidarmanus ORF/protein labelled 1470 is FACI_IFERC00001G1470. Abbreviations: Mja, M. jannaschii; Mth, Methanothermobacter thermautotrophicus; Mka, Methanopyrus kandleri; Mac, M. acetivorans; Hvo, H. volcanii; Pfu, Pyrococcus furiosus; Fac, Ferroplasma acidarmanus; Hs, human; Sc, Saccharomyces cerevisiae; and Um, Ustilago maydis.