DNA replication, DNA damage response and genomic stability
To maintain genome integrity, DNA must be protected from damage occurring during replication or induced by environmental agents. Different cellular mechanisms such us DNA repair, DNA recombination and DNA damage-checkpoints contribute to preserve DNA integrity and cell survival under conditions that produce DNA alterations. We are mainly interested in the DNA-damage checkpoint, and more specifically in the intra S-phase Checkpoint, which is essential to preserve cell viability after replication in the presence of DNA damage. From a number of processes regulated by the DNA-damage checkpoint response, replication fork regulation is one of its major functions and it is the central topic of our research.
The interest of the laboratory is to understand the molecular mechanisms by which the DNA damage-checkpoint preserves replication fork integrity and functionality after encountering DNA lesions during replication. Fork integrity is preserved via inhibition of negative regulators that, otherwise, will lead to loss of fork integrity and incomplete replication. In addition, there are positive regulators required to promote the restart of the replication forks after the lesions/blocks have been eliminated. Our studies are focused on how these regulators are controlled by the checkpoint machinery and the relative contribution of different checkpoint proteins in their regulation.
We study the checkpoint-dependent regulation of identified factors involved in fork stabilization by using genetic and biochemical methods combined with genomic and proteomic approaches, and, moreover, the identification of new factors participating in this complex process is our top priority.
The budding yeast Saccharomyces cerevisiae is the experimental model used in our laboratory. In this yeast, the components of the replication machinery and checkpoints pathways are well characterized and are evolutionary conserved, being a good genetic and biochemical model for the study of DNA replication control in eukaryotes.
A) Schematic of the intra-S-Phase Checkpoint Response.
In the presence of DNA lesions or replication perturbations, the checkpoint kinases Mec1 and Rad53 are activated, and a complex response is triggered which includes the regulation of cell cycle progression, fork stability, late origin activation and DNA repair.
B) Analysis of Replication by density transfer substitution.
This technique allows monitoring fork progression along a chromosomal region, and allows accurate quantification of the percentage of replicated DNA in a population of cells during S phase. Cells were blocked in G1 with a-factor and released into media containing HU for 90 min. After that, cultures were released into HU-free media for 120 min. The position of unreplicated (HH) and replicated (HL) DNA peaks is indicated at the top.
C) Phospho-proteomic analysis of factors involved in fork stability.
|Mónica Segurado||Principal Investigator
|Alberto Bugallo||PhD Student|
|Itziar Guerra||Bachelor Degree Student|
|María Fernández||Laboratory Technician|
| Morafraile EC, Bugallo A, Carreira R, Fernández M, Martín-Castellanos C, Blanco MG, Segurado M (2020)
Exo1 phosphorylation inhibits Exonuclease activity and rescues fork collapse in rad53 mutants independently of the 14-3-3 proteins.
Nucleic Acids Research gkaa054
| Bustamante-Jaramillo LF, Ramos C, Alonso L, Sesmero A, Segurado M, Martín-Castellanos C (2019)
CDK contribution to DSB formation and recombination in fission yeast meiosis.
PLoS Genetics 15(1):e1007876
| Morafraile EC, Diffley JFX, Tercero JA and Segurado M (2015)
Checkpoint-dependent RNR induction promotes fork restart after replicative stress.
Scientific Reports 5 : 7886
| Soriano I, Morafraile EC, Vazquez E, Antequera F And Segurado M (2014)
Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae.
BMC Genomics15: 791
| Saugar I, Vazquez MV, Gallo-Fernandez M, Ortiz-Bazan MA, Segurado M, Calzada A and Tercero JA (2013)
Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage.
Nucleic Acids Research 41: 8943-8958
| Cotobal C, Segurado M and Antequera F (2010)
Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe.
EMBO J. 29: 934-942
| Segurado M. and Diffley JFX (2008)
Separate roles for the DNA damage checkpoint protein kinases in stabilizing DNA replication forks
Genes Dev. 22: 1816-1827
|Junta de Castilla y León||CSI149P20
Unidad de Investigación Consolidada UIC085