The origin recognition complex protein family

Abstract

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.

Figure 1

Comparison of domains for Orc1-5 and Cdc6 from S. cerevisiae. Orc1, Orc4, Orc5, and Cdc6 each contain an AAA+ domain as part of a larger ORC/Cdc6 domain (orange) [75]. Orc2 and Orc3 are predicted to share this domain structure [19], but have a greater degree of sequence divergence. Motifs within the AAA+ domain include Walker A (WA), Walker B (WB), Sensor-1 (S1) and Sensor-2 (S2). The carboxy-terminal region of ORC/Cdc6 is predicted to contain a winged-helix domain (WH), involved in DNA binding. Orc1 contains an additional BAH (bromo-adjacent homology) domain (pink), which interacts with the Sir1 protein and is involved in epigenetic silencing. Orc1 and Orc2 have regions of disorder (yellow); a DNA-binding AT-hook motif (here PRKRGRPRK) is identified in S. cerevisiae Orc2, and several of these have also been identified in disordered regions in S. pombe Orc4. The number of amino acids for each protein is indicated at the right.

Figure 2

Homology between Orc6 in representative species D. melanogaster (Dm), H. sapiens (Hs), A. thaliana (At), S. pombe (Sp), and S. cerevisiae (Sc). Orc6 contains a unique conserved domain, identified by homology with the Orc6 protein fold superfamily (pfam 05460) [76]. This domain is interrupted by a large disordered region [77] in S. cerevisiae. Orc6 has no recognizable homology to Orc1-5 or AAA+ domains. The carboxy-terminal region of Orc6 in D. melanogaster has been shown to interact with a coiled-coil region of the septin protein Pnut, possibly mediated by coiled-coil motifs predicted in Orc6 [78]. The number of amino acids for each protein is indicated at the right.

Figure 3

ORC and its interactions with other pre-RC proteins at origins of DNA replication. Orc1-Orc5 are required for origin recognition and binding in S. cerevisiae, whereas Orc6 is dispensable in this regard [44]. In contrast, Orc6 is essential for ORC DNA binding in D. melanogaster [28]. Studies with both S. cerevisiae and human cells have indicated that Cdc6 interacts with ORC through the Orc1 subunit (indicated by a double arrow) [31,79,80]. This association increases the specificity of the ORC-origin interaction [20]. Further studies with S. cerevisiae suggest that hydrolysis of Cdc6-bound ATP promotes the association of Cdt1 with origins through an interaction with Orc6 (indicated by a double arrow) [25,31], and this in turn promotes the loading of Mcm2-7 helicase onto chromatin.

Figure 4

Neighbor-joining tree for ORC and Cdc6 proteins. Orc1-5 and Cdc6 sequences were retrieved from the NCBI protein database for H. sapiens (Hs), X. laevis (Xl), D. melanogaster (Dm), S. cerevisiae (Sc), and S. pombe (Sp). The protein corresponding to Cdc6 in S. pombe is named Cdc18 in this species. AAA+ domain regions were extracted from Orc1-5 and Cdc6 sequences using the Walker A and Walker B motifs identified in [19]. The multiple sequence alignment program Muscle [81] was used to align the sequences, and any regions in the multiple sequence alignment containing gaps were deleted. The resulting ungapped alignment was used to construct a phylogenetic tree using the BioNJ algorithm [82]. One hundred resampled alignments were used to generate bootstrap values, with values greater than 70% indicated. For the five eukaryotic organisms from yeast to human, the Orc1-5 and Cdc6 sequences are conserved across all organisms. Orc1 seems to be the most highly conserved, and Orc3 the most divergent, within a group. Interestingly, Orc1 is most closely related to Cdc6 within the ORC-Cdc6 family. Orc6 was not aligned, as it does not share the AAA+ domain with the other members. Scale bar represents changes per site.