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Molecular Medicine Programme | Cancer

DNA Repair and Genome Integrity

Miguel González Blanco
Group Leader | Associate Professor
miguel.gonzalez.blanco@usc.es
 
Field of knowledge

From bacteria to elephants, cells must accomplish fundamental tasks like the preservation, duplication and transmission of their genetic information each time they divide. In our lab, we study how cells coordinate the actions of the DNA damage repair machinery with other major molecular events like DNA replication and chromosome segregation. We currently employ a multi-pronged approach that includes biochemistry, yeast genetics and mammalian cell biology to focus on the following questions:

•  how the biochemical properties of Holliday junction resolvases can bias repair towards different genetic outcomes?
•  how post-translational modifications control the biological functions of DNA repair enzymes?
•  what is the cellular relevance of the evolutionarily conserved regulation of structure-selective nucleases by the cell-cycle progression machinery?

Research Lines

  • Biochemical characterization of DNA repair nucleases and helicases.
  • Analysis of cell cycle- and checkpoint-dependent regulation of DNA repair enzymes.
  • Discovery of novel genetic interaction between genome maintenance factors.

Members

 

Vanesa Hurtado Nieves
PhD student
Laura Rivadulla Costa
FPU fellowship
María Crugeiras Ríos
FPU fellowship
Tomás Lama Díaz
PhD student

Selected publications

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

Raquel Carreira, F Javier Aguado, Vanesa Hurtado-Nieves, Miguel G Blanco
Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I
Arter, Meret; Hurtado-Nieves, Vanesa; Oke, Ashwini; Zhuge, Tangna; Wettstein, Rahel; Fung, Jennifer C.; Blanco, Miguel G.; Matos, Joao

Dual control of Yen1 nuclease activity and cellular localization by Cdk and Cdc14 prevents genome instability.

Blanco MG, Matos J, West SC.

Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis.

Matos J, Blanco MG, Maslen S, Skehel JM, West SC.

Identification of Holliday junction resolvases from humans and yeast.

Ip, S., Rass, U., Blanco, M. et al.

Inhibition of DNA synthesis by K+-stabilised G-quadruplex promotes allelic preferential amplification.

Boán F, Blanco MG, Barros P, González AI, Gómez-Márquez J.

Selected Results

1. The cell cycle progression machinery controls Yen1 nuclease activity and cellular localization to prevent genome instability (Blanco et al., 2014, Mol. Cell).

(A) Cdk and Cdc14 control the phosphorylation status of Yen1. In S phase, Cdk phosphorylates Yen1 to (1) promote its nuclear exclusion by impairing its NLS function and, potentially, enhance Msn5-dependent export, and (2) reduce its DNA binding affinity, avoiding the cleavage of S phase-specific DNA structures such as replication or early recombination intermediates. At anaphase, Cdc14 dephosphorylates Yen1 to reinstate a fully functional NLS and potentially block Msn5-mediated export, leading to its nuclear accumulation. Additionally, the removal of phosphate groups increases the DNA binding affinity and catalytic activity of Yen1, allowing it to resolve HR intermediates that have persisted until anaphase. Red circles (P) depict phosphate groups. 
(B) Yen1ON cannot be turned off or shuttled to the cytoplasm by Cdk. This may lead to the unscheduled and detrimental cleavage of replication or early recombination intermediates. In mutants that accumulate HR intermediates, the constitutive activation of Yen1ON provides an alternative way to process these potentially toxic DNA structures. 

 

2. Premature activation of Yen1 alters the spatial distribution of crossover events in meiosis (Arter et al., 2018, Dev. Cell)

CDK-dependent phosphorylation of Yen1 activity prevents the premature conversion of early recombination intermediates into COs (left panel). This allows adequate establishment of CO-interference between CO-designated joint molecules (JM), resulting in evenly spaced COs and high spore viability. Expression of the constitutively active mutant Yen1ON (right panel) results in premature CO formation and loss of local interference. This enables the CO-designation of neighbouring JMs, leading to uneven CO distribution and reduced spore viability. 
 

3. Biochemical analysis of Yen1 reveals both canonical and non-canonical modes of HJ resolution (Carreira et al., 2022, Nucl. Acids Res.)

Yen1 is able to process fully-ligated Holliday junctions into two nicked duplex DNA molecules using a canonical resolution mechanism (top). Alternatively, a two-step reaction termed dubbed “arm chopping” (bottom) that involves a three-way, replication fork-like intermediate yields a nicked DNA molecule and two smaller DNA duplex molecules, thus resulting in the formation a new double-strand break. Such behaviour is conserved to some extent in other members of the class IV Rad2/XPG-family of nucleases.

Projects

National project(s)

Development of a first-in-class targeted therapy for metastatic NSCLC.
REF: Ref: LABAE211721DELADuration: -
PI: María de la Fuente Freire
Regulación de resolvasas en el procesado de estructuras secundarias del ADN - GENERACIÓN DE CONOCIMIENTO 2020
REF: PID2020-115472GB-I00Duration: -
PI: Miguel González Blanco
AGENCIA ESTATAL DE INVESTIGACION