Faculty Opinions recommendation of Sequence-independent DNA binding and replication initiation by the human origin recognition complex.

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

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Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

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Subsets of Human Origin Recognition Complex (ORC) Subunits Are Expressed in Non-proliferating Cells and Associate with Non-ORC Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m005765200 ◽

2000 ◽

Vol 275 (45) ◽

pp. 35233-35241 ◽

Cited By ~ 30

Author(s):

Kelly C. Thome ◽

Suman K. Dhar ◽

David G. Quintana ◽

Laurie Delmolino ◽

Aliakbar Shahsafaei ◽

...

Keyword(s):

Origin Recognition Complex ◽

Human Origin ◽

Proliferating Cells ◽

Origin Recognition

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

Journal of Biological Chemistry ◽

10.1074/jbc.m113.492504 ◽

2013 ◽

Vol 288 (42) ◽

pp. 30161-30171 ◽

Cited By ~ 53

Author(s):

Shoko Hoshina ◽

Kei Yura ◽

Honami Teranishi ◽

Noriko Kiyasu ◽

Ayumi Tominaga ◽

...

Keyword(s):

Origin Recognition Complex ◽

Human Origin ◽

Single Stranded Dna ◽

G Quadruplex ◽

Origin Recognition

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Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA

Nucleic Acids Research ◽

10.1093/nar/gkaa751 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11146-11161

Author(s):

Naining Xu ◽

Yingying You ◽

Changdong Liu ◽

Maxim Balasov ◽

Lee Tung Lun ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Solution Structure ◽

Origin Recognition Complex ◽

Structural Basis ◽

Dna Replication Origin ◽

Dna Alterations ◽

Replication Initiator ◽

Binding Subunit ◽

Origin Recognition

Abstract The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

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Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Journal of Biological Chemistry ◽

10.1074/jbc.m110.215988 ◽

2011 ◽

Vol 286 (27) ◽

pp. 23831-23841 ◽

Cited By ~ 19

Author(s):

Soma Ghosh ◽

Alex P. Vassilev ◽

Junmei Zhang ◽

Yingming Zhao ◽

Melvin L. DePamphilis

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Origin Recognition Complex ◽

Genome Duplication ◽

Cyclin Dependent Kinase ◽

Human Origin ◽

Cell Extracts ◽

Localization Signal ◽

Origin Recognition

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2–5) complex in the nucleus, an ORC(1–5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1–6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2–5) are transported independently to the nucleus where they can either assemble into ORC(1–6) or function individually.

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