Structural insights into the Cdt1-mediated MCM2–7 chromatin loading
Nucleic Acids Research
Structural insights into the Cdt1-mediated MCM2-7 chromatin loading
Changdong Liu 0
Rentian Wu 0
Bo Zhou 0
Jiafeng Wang 0
Zhun Wei 0
Bik K. Tye 1
Chun Liang 0
Guang Zhu 0
0 Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology , Clear Water Bay, Kowloon, Hong Kong , China
1 Department of Molecular Biology & Genetics, Cornell University , USA
Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.
INTRODUCTION
To maintain genome integrity, DNA replication in
eukaryotic cells is tightly regulated to ensure that the
genome is replicated exactly once per cell cycle. This
regulation is achieved through a two-step mechanism, the
loading of the replicative DNA helicase, which includes
the MCM2-7 complex as a major component and
activation of this helicase (
1
).
The loading of the MCM2?7 complex requires the
coordinated action of several proteins, most notably the
six-subunit origin recognition complex (ORC), the cell
division cycle 6 homolog (Cdc6), the chromatin licensing
and DNA replication factor 1 (Cdt1) (
2,3
). The first step
of this process is the binding of ORC to the replication
origin on newly synthesized chromatin followed by the
recruitment of Cdc6 and Cdt1. These two factors then
recruit the MCM2?7 complex to form a prereplicative
complex (pre-RC) during the late M and G1 phases
(
4,5
). Activation of the pre-RC requires the sequential
assembly of additional factors including Cdc45 and the
GINS complex in a DDK- and S-CDK-dependent
manner, culminating in the initiation of DNA replication
in S-phase (
6,7
).
The MCM2?7 complex was first identified as a family of
genes required for minichromosome maintenance in
Saccharomyces cerevisiae (
8
). All six paralogous MCM
proteins belong to the highly diversified AAA+
(ATPases associated with a variety of cellular activities)
protein family (
9
). Structural analysis showed that the six
MCM proteins form a double hexameric ring with
head-to-head configuration, and DNA passes through
the central channel of the double hexamer (
10?12
). The
hexamer shows weak helicase activity in vitro (
13?15
).
The licensing factor Cdt1 is a critical component of the
pre-RC, and its primary function is to recruit the MCM2?
7 complex to the replication origin (
16
). Overexpression of
Cdt1 alone in many types of mammalian cell lines causes
rereplication of DNA (
17?19
).
Recently results showed that the Cdt1 MCM2?7
hepatmer is loaded onto DNA cooperatively to form a
double hexamer (
12
). Previous studies on the interactions
between Cdt1 and individual members of the MCM2?7
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
complex showed that Cdt1 interacts with Mcm2 and
Mcm6 (
16,20?22
). The region of Cdt1 involved in the
MCM2?7 interaction has been defined. Cdt1 binds to
MCM2?7 complex through the region spanning residues
447?620 in Xenopus (16) and residues 407?477 of Cdt1
bind to Mcm6 in mouse (
20
). On the basis of the yeast
two-hybrid assay we found that the interaction between
human Mcm6 and Cdt1 is much stronger than that
between Mcm2 and Cdt1. We further demonstrated that
the conserved C-terminal domain of the human Mcm6
(residues 707?821) physically interacts with Cdt1
(residues 410?445) (
23
). However, the detailed molecular
mechanism underlying the chromatin loading of the
MCM2?7 complex through Cdt1 remains elusive.
In the present study, we determined the solution
complex structure of the Cdt1-Mcm6 binding domains,
the C-terminal helix (411?440) of Cdt1 binds to the
C-terminal region (708?821) of Mcm6. In vivo studies in
S. cerevisiae showed that interruption of this interaction
prevented the (...truncated)