Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB

Abstract

The Origin Recognition Complex (ORC) is a six-subunit protein important for the initiation of DNA replication in eukaryotic cells. Orc6 is the smallest and the least conserved among ORC subunits. It is required for the DNA replication but also has a function in cytokinesis in metazoan species, however, the mechanisms of Orc6 action in these processes are not clear. Here we report a structure of the middle domain of human Orc6. This domain has an overall fold similar to the corresponding helical domain of transcription factor TFIIB. Based on these findings, a model of Orc6 binding to DNA is produced. We have identified amino acids of Orc6 which are directly involved in DNA binding. Alterations of these amino acids abolish DNA binding ability of Orc6 and also result in reduced levels of DNA replication in vitro and in cultured cells. Our data indicate that Orc6 is one of the DNA binding subunits of ORC in metazoan species. We propose that Orc6 may participate in positioning of ORC at the origins of DNA replication similar to the role of TFIIB in positioning transcription preinitiation complex at the promoter.

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

“…However, these results do not necessarily mean that the same inhibition occurs in vivo, because the length of probes/competitors used in this study is limited. It has been reported that ORC4 has an affinity to DNA triplex (43) and that ORC6 has a TFIIB-like DNA-binding domain (44). Thus, it may be possible that the entire ORC binds simultaneously to dsDNA and RNA/ssDNA if an appropriate substrate is used.…”

Nonreplicative functions of the origin recognition complex

“…The ORC6 protein possesses two functional domains. The conservative N-terminal domain is responsible for DNA interaction, and in metazoans shows structural homology with the transcription factor TFIIB [100]. A small variable domain at the C-terminus is responsible for interactions with other proteins, and with ORC3, in particular [20,101,102].…”

Helicase Loading at Chromosomal Origins of Replication

“…Four ORC subunits (Orc1, -2, -4, and -5) also contain potential wingedhelix domains at their carboxyl termini. The smallest ORC subunit, Orc6, is related to the RNA Pol II auxilliary factor TFIIB in metazoans (Liu et al 2011), although fungal Orc6 lacks this similarity. Like Mcm2 -7 and ORC, Cdc6 is an AAA þ protein in the same initiator clade as the AAA þ ORC subunits, DnaA and DnaC (Iyer et al 2004).…”