Two subunits of human ORC are dispensable for DNA replication and proliferation

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication

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Architecture of the yeast origin recognition complex bound to origins of DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7159 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7159-7168 ◽

Cited By ~ 127

Author(s):

D G Lee ◽

S P Bell

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Origin Recognition Complex ◽

Binding Assays ◽

Chromosomal Replication ◽

Origins Of Replication ◽

Eukaryotic Dna ◽

Level Of Analysis ◽

Origin Recognition

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.

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HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00506-19 ◽

2019 ◽

Vol 40 (4) ◽

Author(s):

Andrew J. Kueh ◽

Samantha Eccles ◽

Leonie Tang ◽

Alexandra L. Garnham ◽

Rose E. May ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Adhesion ◽

Dna Replication ◽

Cell Lines ◽

Human Cell ◽

Human Cells ◽

Human Cell Lines ◽

Hela Cell Lines ◽

Mouse Tissues ◽

Sirna Knockdown

ABSTRACT HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.

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Rethinking origin licensing

eLife ◽

10.7554/elife.24052 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 5

Author(s):

Stephen P Bell

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

Human Cells ◽

Origin Licensing ◽

A Subunit ◽

Origin Recognition

Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication.

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