Ir o contido principal
Programa de Mecanismos Moleculares | Cancro

DNA Repair and Genome Integrity

Miguel González Blanco
Miguel González Blanco
Group Leader | Associate Professor
miguel.gonzalez.blanco@usc.es
ORCID: 0000-0002-2883-7326
 
Área de coñecemento

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?

Liñas de investigación

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

Membros

 

Laura Rivadulla Costa
PhD student, FPU Fellowship
María Crugeiras Ríos
PhD student, FPU Fellowship
Clara Miranda Abeijón
PhD student, Xunta de Galicia Fellowship
Lucía Debasa Corral
PhD student

Publicacións seleccionadas

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

Carreira R, Lama-Diaz T, Crugeiras M, Aguado FJ, Sebesta M, Krejci L, Blanco MG.
. Nucleic Acids Res. 2024 Jul 8;52(12):7012-7030.

Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase.

Lama-Diaz T, Blanco MG
Nucleic Acids Research. 2024 May 23:gkae400

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

Carreira R, Aguado FJ, Hurtado-Nieves V, Blanco MG
Nucleic Acids Research, Volume 50, Issue 1, 11 January 2022, Pages 259?280

Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I

Meret A, Hurtado-Nieves V, Oke A, Zhuge T, Wettstein R, Fung, JC, Blanco, MG, Matos J.
DEVELOPMENTAL CELL, 2018 Jun; 45 (6): 785+

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

Blanco MG, Matos J, West SC.
Mol Cell. 2014 Apr 10;54(1):94-106.

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

Matos J, Blanco MG, Maslen S, Skehel JM, West SC.
Cell. 2011 Sep 30;147(1):158-72.

Identification of Holliday junction resolvases from humans and yeast.

Ip, S., Rass, U., Blanco, M. et al.
Nature volume 456, pages357–361 (2008).

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

Boán F, Blanco MG, Barros P, González AI, Gómez-Márquez J.
FEBS Lett. 2004 Jul 30;571(1-3):112-8.

Resultados seleccionados

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.

 

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

Using a biochemical system for the generation of D-loops in vitro employing purified Rad51, Rda54 and RPA, we demonstrated that the half-crossover (top) and chromosomal loss (bottom) events observed in genetic assays for break-induced replication after expression of de-regulated Yen1 (Elango et al., 2017) can be explained by the incisions produced by the Mus81-Mms4 and Yen1 on these structures. Moreover, the appearance of bona-fide half-crossover precursors is detected in our system upon addition of a DNA ligase, constituting the first report to our knowledge of the recreation of this exotic recombination product in th test tube.

 

5. Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase (Lama-Diaz and Blanco, 2024, Nucleic Acids Res.)

In multiple organisms, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability.

 

Proxectos

Proxecto(s) activo(s)

Proxectos nacionais

Identificación de motivos funcionales en el dominio N-terminal de helicasas de la familia de Pif1- GENERACIÓN DE CONOCIMIENTO 2023
REF: PID2023-147024NB-I00Duration: - AGENCIA ESTATAL DE INVESTIGACION
CONSOLIDACIÓN E ESTRUTURACIÓN 2023 GRC GI-2119 - Mecanismos Moleculares de la Enfermedad - MeMoEn
REF: ED431C 2023/10Duration: -
PI: Juan Bautista Zalvide Torrente
AXENCIA GALEGA DE INNOVACION (GAIN)

Proxecto(s) finalizado(s)

Proxectos nacionais

Development of a first-in-class targeted therapy for metastatic NSCLC.
REF: Ref: LABAE211721DELADuration: -
PI: María de la Fuente Freire
Ver más información

Funder: Fundación Científica Asociación Española contra el Cáncer.

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