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Vice-Direction of Basic Research
DNA REPLICATION GROUP
Molecular Oncology Programme | DNA Replication Group
Juan Méndez
Group Leader
Post-Doctoral Fellow
Sara Rodríguez
Graduate Students
Marcos Díaz, Daniel González,
Karolina Jodkowska, Sergio Muñoz
OVERVIEW
Our laboratory studies the process of DNA replication. All cells
that proliferate synthesise a replica of their genome before
undergoing mitotic division. DNA replication makes the genome
vulnerable to mutations and chromosomal reorganisations, and
constitutes one of the unavoidable risk factors in developing
cancer. On the other hand, as cancer cells are highly proliferative,
the proteins that synthesise DNA are useful targets for
chemotherapeutic drugs. Our current research interests are :
( 1 ) the control of DNA replication through the regulation of
replication origins ( the genomic positions where ‘ replisomes ’
are assembled to start the synthesis of new DNA ); ( 2 ) the study
of specific proteins that mediate replication of damaged DNA,
such as the primase-polymerase PrimPol ; ( 3 ) the consequences
of deregulated replication in cancer and ageing. In our different
projects we use a combination of technical approaches including
biochemistry, molecular biology and mouse genetics.
ANNUAL REPORT 2015
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SPANISH NATIONAL CANCER RESEARCH CENTRE, CNIO
“ We have found that certain blood
cell types are highly sensitive to
replication stress. A partial loss
of the replicative DNA helicase
affects haematopoietic stem cells
and erythroblasts, causing anaemia
and increasing the frequency of
haematological cancers.”
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Vice-Direction of Basic Research
Molecular Oncology Programme | DNA Replication Group
RESEARCH HIGHLIGHTS
Figure 2 Cdc6 deregulation affects
the hair growth cycle. Distribution of
wild-type and K5-CDC6 mice (>105
weeks old ) in 3 phenotypic categories
according to their fur preservation.
Figure 1 Impaired red blood cell
production in Mcm3-deficient mice.
( A ) Competitive transplantation ( 1 :1 )
between Mcm3-deficient foetal liver
cells and Mcm3-competent bone
marrow cells expressing fluorescent
Lethal anaemia caused by hypomorphic expression of
MCM helicase
We have shown, using a mouse strain with hypomorphic
expression of the Mcm3 gene, that limiting the number of
potential replisomes in vivo affects the functionality of
haematopoietic stem cells and the differentiation of rapidlydividing erythrocyte precursors ( FIGURE 1, A and B ). In
addition, the lifespan of Mcm3-hemizygous mice is reduced
due to early-onset lymphomas and mesenchymal tumours.
When the concentration of MCM3 protein becomes <1/3 of its
normal levels, embryos die in utero because the foetal liver fails
to make enough red blood cells to sustain oxygen delivery to all
tissues. During the last year, we demonstrated the link between
defective DNA replication and the anaemic phenotype. As
erythrocyte precursor cells maturate in the liver, they undergo
several rounds of rapid cell division that require a dynamic
programme of DNA replication. In the last proliferation cycles
of normal maturing erythroblasts, the speed of the replication
machinery progressively decreases and, in turn, more origins
are activated to complete replication before reaching mitosis.
Mcm3-deficient erythroblasts cannot follow this programme
and become blocked at the late basophilic stage, preventing the
ANNUAL REPORT 2015
CDC6 overexpression extended the
resting stage of hair follicles, increasing
hair preservation. Adapted from Búa
et al. ( 2015 ).
Tomato protein. ( B ) Red blood cell
chimerism in recipient mice after
transplantation. ( C ) Accumulation
of immature erythroblasts in Mcm3deficient peripheral blood. Adapted
from Alvarez et al. ( 2015 ).
formation of mature red blood cells and causing a lethal form of
aplastic anaemia ( FIGURE 1, C ). One interesting aspect of this
study is that viability can be rescued by introducing an extra
copy of checkpoint kinase Chk1, which alleviates replicative
stress in other contexts ( López-Contreras et al., J Exp Med,
2012 ). In collaboration with the Genomic Instability Group, we
found that >50% of MCM3-deficient embryos carrying higher
levels of Chk1 completed gestation and survived as adults with
a mild anaemia ( Alvarez et al., 2015 ). This result opens up the
interesting challenge of enhancing the cellular response to
replicative stress as a means to counteract aplastic anaemias
that are frequently associated with chemotherapy treatments.
Cdc6 overexpression affects papillomagenesis and
influences hair growth
In 2015, we also completed a study to monitor the effect of Cdc6
deregulation in vivo. Cdc6 encodes a protein responsible for
the recruitment of MCM helicase to replication origins and
is overexpressed in several cancer types, including subsets of
brain tumours, mantle cell lymphomas and non-small cell lung
carcinomas. To date, no model has been described to test the
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Applications of single-molecule analysis of DNA
replication
proto-oncogenic effects of Cdc6 deregulation in mammalian
tissues. The K5-Cdc6 mice strain generated at the CNIO displayed
higher levels of CDC6 protein in the skin and other tissues with
stratified epithelia. Cdc6 “ gain of function ” was revealed by
the enhanced loading of MCM complexes in keratinocytes.
Deregulated Cdc6 by itself did not promote skin tumours, but
in combination with chemical carcinogens it favoured the
formation of benign papillomas. Furthermore, older K5-CDC6
mice displayed better fur preservation than their wild-type
littermates ( FIGURE 2 ). This unanticipated ‘ anti-ageing ’ effect
was analysed in collaboration with the laboratory of C. Blanpain
( Université Libre de Bruxelles, Belgium ); we found that CDC6
extended the resting stage of the hair follicle growth cycle ( Bua
et al., 2015 ).
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PUBLICATIONS
Alvarez S, Díaz M, Flach J, Rodríguez-Acebes S, López-Contreras A, Martínez D,
Cañamero M, Fernández-Capetillo O,
Isern J, Passegué E, Méndez J ( 2015 ).
Replication stress caused by low MCM
expression limits fetal erythropoiesis
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and hematopoietic stem cell functionality. Nat Commun 6, 8548. DOI : 10.1038/
ncomms9548.
Jacome A, Gutiérrez-Martínez P, Schiavone
F, Tenaglia E, Martínez P, Rodríguez-Acebes S, Lecona E, Murga M, Méndez J, Blasco MA, Fernández-Capetillo O ( 2015 ).
NSMCE2 suppresses cancer and ageing
SPANISH NATIONAL CANCER RESEARCH CENTRE, CNIO
One of our approaches for the study of DNA replication consists
of the analysis of replisome progression and origin activity in
individual DNA molecules. To this end, we extensively use a
‘ stretched DNA fibre ’ technique that has attracted the interest of
other CNIO Research Groups. In 2015, single-molecule analysis
of DNA replication was applied to several projects, including
the study of megakaryocyte polyploidisation mechanisms led
by Marcos Malumbres ( Trakala et al., 2015 ) and the study
of NSMCE3 in cancer and ageing, led by Oscar FernandezCapetillo ( Jacome et al., 2015 ). We are currently working on the
development of novel applications for this powerful technique. s
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in mice independently of its SUMO ligase
activity. EMBO J 34, 2604-2619.
Trakala M, Rodríguez-Acebes S, Maroto
M, Symonds KE, Santamaría D, Ortega
S, Barbacid M, Méndez J, Malumbres M
( 2015 ). Functional reprogramming of
polyplodization in megakaryocytes. Dev
Cell 32, 155-167.
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Búa S, Sotiropoulou P, Sgarlata C, Borlado, LR, Eguren M, Domínguez O, Ortega
S, Malumbres M, Blanpain C, Méndez J
( 2015 ). Deregulated expression of Cdc6
in the skin facilitates papilloma formation
and affects the hair growth cycle. Cell
cycle 14, 3897-3907.
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