Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Mar 16;96(6):2656-61.
doi: 10.1073/pnas.96.6.2656.

The Fission Yeast Homologue of Orc4p Binds to Replication Origin DNA via Multiple AT-hooks

Affiliations
Free PMC article

The Fission Yeast Homologue of Orc4p Binds to Replication Origin DNA via Multiple AT-hooks

R Y Chuang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae.

Figures

Figure 1
Figure 1
Identification of Orp4. (A) Diagram of the sequence organization of the Orp4 protein. The locations of the nine AT-hooks in the N-terminal domain are indicated by vertical bars. (B) Alignment of the sequences of the AT-hook motifs in Orp4p. (C) Predicted amino acid sequence of the Orp4 open reading frame. The nine AT-hooks in the N-terminal domain are shaded and the putative ATP/GTP binding motifs in the C-terminal domain are underlined. The region of the protein with homology to human and yeast Orc4 proteins is boxed.
Figure 2
Figure 2
Sequence alignment of Orc4 proteins. The amino acid sequence of the C-terminal domain of Orp4 protein (sporp4) is aligned with the sequences of the Orc4 proteins of human (hsorc4), frog (xlorc4), and budding yeast (scorc4). Identical amino acids shared by the majority of sequences are shaded black and similar amino acids are shaded gray. The Walker A and B sites are indicated.
Figure 3
Figure 3
Orp4 protein is expressed throughout the cell cycle. Fission yeast cells expressing epitope-tagged Orp4 protein were blocked in the G1, S, G2, or M phases of the cell cycle as described in Materials and Methods. Extracts prepared from blocked cells were fractionated by SDS/PAGE and Orp4 protein was detected by Western blot analysis. The same Western blots were probed for tubulin protein as a control for equal loading of the gels.
Figure 4
Figure 4
Phenotypic effects of deletion or over-expression of Orp4 protein. (A and B) Deletion of Orp4. Spores from a control diploid strain (ura4+/ura4-D18) or a diploid strain in which one copy of the orp4+ gene replaced by the ura4+ gene (ura4-D18/ura4-D18 orp4+/orp4∷ura4+) were incubated in medium lacking uracil for 15 hr and stained with DAPI. The control spores yielded a population of growing vegetative cells with mostly normal morphology (A). Spores lacking the orp4+ gene yielded a variety of terminal phenotypes, including elongated cells with abnormal nuclei (n) and “cut” cells (c) with the septum separating unequal nuclear masses (B). An ungerminated spore is marked by (s). (C–F) Over-expression of Orp4 protein or its domains. Wild-type Sc. pombe cells were transformed with vector alone (C) or with plasmids expressing the N-terminal domain of Orp4p (D), the C-terminal domain of Orp4p (E) or the complete Orp4p (F) under the control of the inducible nmt1+ promoter. Twenty hours after induction the cells were fixed and stained with DAPI. Approximately 30% of cells over-expressing the N-terminal domain of Orp4p contained a septum with a single nucleus (D). Arrows indicate septa. The majority of cells over-expressing the C-terminal domain of Orp4p were elongated and many exhibited abnormal nuclear morphology (E). Cells over-expressing the complete Orp4p showed both phenotypes (F).
Figure 5
Figure 5
Binding of Orp4 protein to the ars1 replication origin. Cell extracts were prepared from haploid cells expressing full length Orp4p (F), the N-terminal domain of Orp4p (N), or the C-terminal domain of Orp4p (C). Extracts from cells containing vector alone (V) served as a control. After SDS/PAGE the cellular proteins were transferred to a nitrocellulose membrane and subjected to a renaturation protocol. The membranes were incubated with a radiolabeled fragment of the ars1 replication origin in the presence or absence of a 100-fold excess of nonradioactive competitor DNA. The competitor DNAs consisted of the identical ars1 fragment (specific) or an unrelated fragment of similar size (nonspecific). The positions of full-length Orp4p, the N-terminal domain and the C-terminal domain are indicated as F, N, and C, respectively. (The expression of all three proteins was verified by Western blot analysis.) The position of endogenous Orp4p is indicated by E.

Similar articles

See all similar articles

Cited by 98 articles

See all "Cited by" articles

Publication types

Associated data

Feedback