The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein
Jk. Lee et al., The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein, P NAS US, 98(24), 2001, pp. 13589-13594
Citations number
32
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
The interaction between an origin sequence and the origin recognition compl
ex (ORC), which is highly conserved in eukaryotes, is critical for the init
iation of DNA replication. In this report, we have examined the interaction
between the Schizosaccharomyces pombe (sp) autonomously replicating sequen
ce 1 (ars1) and the spORC. For this purpose, we have purified the spORC con
taining all six subunits, a six-subunit complex containing the N-terminal-d
eleted spOrc4 subunit (spORC(DeltaN-Orc4)), and the spOrc4 subunit by using
the baculovirus expression system. Wild-type spORC showed sequence-specifi
c binding to arsi, and the spOrc4 protein alone showed the same DNA-binding
properties as wild-type spORC. In contrast, the spORC(DeltaN-Orc4) and the
DeltaN-spOrc4p alone did not bind significantly to ars1. These findings in
dicate that the N-terminal domain of the spOrc4 protein that contains multi
ple AT-hook motifs is essential for the ars1-binding activity. DNA-binding
competition assays with fragments of ars1 and DNase I footprinting studies
with full-length ars1 revealed that the spORC interacted with several AT-ri
ch sequence regions of ars1. These DNA-binding properties of spORC correlat
e with the previously determined sequence requirements of the S. pombe ars1
. These studies indicate that because of its unique Orc4 subunit, S. pombe
uses a mechanism to recognize its origins different from that used by Sacch
aromyces cerevisiae.