Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of 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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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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Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

Journal of Cell Science ◽

10.1242/jcs.01405 ◽

2004 ◽

Vol 117 (22) ◽

pp. 5221-5231 ◽

Cited By ~ 38

Author(s):

M. R. Lidonnici

Keyword(s):

Cell Cycle ◽

Origin Recognition Complex ◽

Human Origin ◽

Origin Recognition

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The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.4.4.832-835.2005 ◽

2005 ◽

Vol 4 (4) ◽

pp. 832-835 ◽

Cited By ~ 13

Author(s):

Terri S. Rice ◽

Min Ding ◽

David S. Pederson ◽

Nicholas H. Heintz

Keyword(s):

Saccharomyces Cerevisiae ◽

Origin Recognition Complex ◽

Nuclear Division ◽

Permissive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

M Phase ◽

And Migration ◽

Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

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Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2790-2801.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2790-2801 ◽

Cited By ~ 31

Author(s):

James F. Theis ◽

Carol S. Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Binding Site ◽

Binding Sites ◽

Origin Recognition Complex ◽

Replication Origins ◽

Discrete Site ◽

Origin Function ◽

Single Binding Site ◽

Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

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