Interaction Of Origin Recognition Complex Research Articles

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress.

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila.

The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

Different roles of the human Orc6 protein in the replication initiation process.

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1(27-149)]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1(27-149) in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

Structure of the Sir3 protein bromo adjacent homology (BAH) domain from S. cerevisiae at 1.95 Å resolution

Sir3p is a silent-information-regulator (SIR) protein required for the assembly of a transcriptionally "silent" chromatin structure at telomeres and the cryptic HM mating-type loci in Saccharomyces cerevisiae. Sir3p contains a putative "bromo adjacent homology" (BAH) domain at its N terminus that shares strong sequence similarity with the BAH domain of a subunit of the origin recognition complex (ORC), Orc1p. The Orc1p-BAH domain forms a well-defined complex with the ORC interaction region (OIR) of another Sir protein, Sir1p, which targets formation of silent chromatin to the HM-loci. Interestingly, despite sequence similarity of the Sir3p and Orc1p BAH domains and Sir3p's established importance in silencing, Sir3p does not bind the Sir1p-OIR. Here we report the 1.95 A resolution crystal structure of the Sir3p-BAH domain. The structure reveals two key features that can account for Sir3p-BAH domain's inability to interact with Sir1p. First, several Orc1p-BAH domain residues known to directly contact Sir1p are altered in the Sir3p-BAH domain. Second, a critical OIR-binding pocket present on the surface of the Orc1p-BAH domain is "filled" in the Sir3p-BAH domain structure, potentially making it inaccessible to Sir1p. These findings imply that the Sir3p-BAH domain structure has evolved for functions distinct from those of the Orc1p-BAH domain.

Structural basis of the Sir1–origin recognition complex interaction in transcriptional silencing

The Sir1 protein plays a key role in establishing a silent chromatin structure at the cryptic mating-type loci HMR and HML in Saccharomyces cerevisiae by interacting with the bromo-adjacent homology (BAH) domain of the Orc1p subunit of the origin recognition complex (ORC). Here, we present the high-resolution crystal structures of the ORC interaction region (OIR) of Sir1p and that of the complex formed between the OIR and BAH domains. Amino acids within the OIR previously shown to be required for a Sir1p/ORC interaction are presented on a conserved, convex surface that forms a complementary interface with a concave region of the Orc1 BAH domain that is critical for transcriptional silencing. The OIR/BAH interaction surface comprises a network of hydrophobic and polar/ionic interactions between discrete structural modules in each protein and involves several residues that were not implicated in previous studies. These data provide important structural insights into a protein-protein interaction critical for the formation of a specialized chromatin domain within eukaryotic chromosomes.

The origin recognition complex and Sir4 protein recruit Sir1p to yeast silent chromatin through independent interactions requiring a common Sir1p domain.

Sir1p is one of four SIR (silent information regulator) proteins required for silencing the cryptic mating-type locus HMRa in the budding yeast Saccharomyces cerevisiae. A Sir1p interaction with Orc1p, the largest subunit of the origin recognition complex (ORC), is critical for Sir1p's ability to bind HMRa and function in the formation of silent chromatin. Here we show that a discrete domain within Sir1p, the ORC interaction region (OIR), was necessary and sufficient for a Sir1p-ORC interaction. The OIR contains the originally defined silencer recognition-defective region as well as additional amino acids. In addition, a Sir1p-Sir4p interaction required a larger region of Sir1p that included the OIR. Amino acid substitutions causing defects in either a Sir1p-Orc1p or a Sir1p-Sir4p interaction reduced HMRa silencing and Sir1p binding to HMRa in chromatin. These data support a model in which Sir1p's association with HMRa is mediated by separable Sir1p-ORC and Sir1p-Sir4p interactions requiring a common Sir1p domain, and they indicate that a Sir1p-ORC interaction is restricted to silencers, at least in part, through interactions with Sir4p.

Metazoan Origin Selection: ORIGIN RECOGNITION COMPLEX CHROMATIN BINDING IS REGULATED BY CDC6 RECRUITMENT AND ATP HYDROLYSIS

Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.

Differential DNA affinity specifies roles for the origin recognition complex in budding yeast heterochromatin.

The origin recognition complex (ORC) marks chromosomal positions as replication origins and is essential for replication initiation. At a few loci, the ORC functions in heterochromatin formation. We show that the ORC's two roles at the heterochromatic HMRa locus in Saccharomyces cerevisiae were regulated by differences in the ORC's interaction with its target site. At HMRa, a strong ORC-DNA interaction inhibited and delayed replication initiation but promoted heterochromatin formation, whereas a weak ORC-DNA interaction allowed for increased and earlier replication initiation but reduced heterochromatin formation. Therefore, the ORC's interaction with its target site could modulate ORC activity within a heterochromatin domain in vivo.

Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle.

Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the S phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G(1)-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.

The Sir1 protein's association with a silenced chromosome domain.

Silencing of the cryptic mating-type locus HMR requires recognition of a small DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins required for the assembly of silenced chromatin domains in Saccharomyces cerevisiae. The Sir1p recognizes the silencer through interactions with the origin recognition complex (ORC), a protein complex that binds the silencer DNA directly. Sir1p was physically associated with HMR in chromatin, and this association required a Sir1p-ORC interaction, suggesting that it reflected the Sir1p silencer-recognition function required for silencing. Sir1p was not associated with nonsilencer replication origins that bind the ORC, indicating that a Sir1p-ORC interaction is confined to silencers. Significantly, the other SIR genes were required for Sir1p's association with HMR. Thus, multiple protein contacts required for and unique to silent chromatin may confine a Sir1p-ORC interaction to silencers. The Sir1p was present at extremely low concentrations in yeast cells yet was associated with HMR at all stages of the cell cycle examined. These data provide insights into the mechanisms that establish and restrict the assembly of silenced chromatin to only a few discrete chromosomal domains.