DNA Replication & Repair: evolution, mechanisms and applications
DNA is the molecule of life. It is depositary of the genetic information of most living beings. The double helix carries the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses. Thus, the evolution shaped mechanism for maintaining the quality of that information through generations. DNA replication assures the transmission of the information to the next generation. Before, during and after replication, there are also processes that check the integrity of DNA and, if required, our cells are endowed with DNA repair pathways for fixing errors.
In our lab, we focus on the protein factors (enzymes) that carry out that work. DNA polymerases are the main responsible for copying the information stored in each DNA strand. Other enzymes, such as DNA Glycosylases or AP endonuclease can surgically identify and remove damage from the double helix.
We use simple genetic models, like transposons, virus and bacteria to analyze the molecular mechanisms that maintain genetic information in the DNA molecule. This also allows us to devise new methods and biotechnological applications. We are currently focused in DNA polymerases but new projects are being cooked under low heat.
You can learn more information about what we do here.
Our funded research grants
Primer-independent DNA polymerases group (piPolBs) constitute a third, previously overlooked, clade of family B DNA polymerases (PolBs), along with the RNA-primed (rPolBs) and protein-primed (pPolBs). PiPolBs are encoded by a new group of mobile genetic elements, named pipolins, most of which are integrated into genomes of bacteria from phyla Firmicutes, Actinobacteria, and Proteobacteria, but also replicating as circular plasmids in mitochondria.
Initial biochemical characterization of piPolBs showed that they share some properties with other replicative DNA polymerases, like a proofreading capacity coupled with processive and faithful DNA polymerization activity. Strikingly, they are also capable of primer-independent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. A modify extended KxY motif, KH-X8-TGR, is involved in this relevant capacity.
Moreover, piPolBs do not display strong sequence requirement for replication initiation and replication origins seem to be selected in a random manner, a property that may be useful for the development of unbiased whole-genome amplification.
In this project, we propose the combined use of bioinformatics, genetic and biochemistry methods to analyze in detail the properties of piPolBs by mutagenesis of clade-specific conserved residues. A hybrid phylogenetic analysis, using a combination of MSA and structural comparisons, will be used to perform a comprehensive classification of PolBs in databases and select new candidates with unforeseen properties for biochemical characterization. In addition, we will also analyze pipolin replication in vivo.
Furthermore, besides providing new insights into the evolution and molecular mechanisms of DNA replication, we will contribute to the developing of novel biotechnology applications for primer-independent DNA polymerases, analyzing whole genome amplification coverage by next-generation sequencing methods for single-molecule DNA amplification using both, piPolB and new candidates of interest detected during the project.
2019-2021 (Fundación Ramón Areces)
In collaboration with professor Margarita Salas, we aim to perform a comprehensive virus-host protein interactome by the use of yeast-two-hybrid coupled to next-generation sequencing analysis. We will use B. thuringienis virus Bam35. You can find our previous work on Bam35 here and here.
This project is a follow up of our previous work on the intraviral interactome, which allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of betatectiviruses.
NewPols4Biotech (in Spanish here), was a research grant from ComFuturo Program. The ultimate goal of this project was developing new tools for DNA amplification that increase the capacity of currently available kits or provide new features for particular needs in different fields, such as archaeology, forensic medicine, personalized treatments, etc.
Thanks to this grant, we could identify and characterize a new group of DNA polymerase, dubbed pipolBs, endowed with DNA priming capacity.