New Mechanisms of DNA replication and Repair
Characterization of catalitic residues of piPolBs, a new group of family B DNA polymerases.
September 2020
Primer-independent DNA polymerases group (piPolBs) constitutes 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.
Analysis of multiple sequence alignments (MSAs) of piPolBs showed that all of them contained active site residues of the exonuclease and DNA polymerase activities of PolBs, albeit with notable variations within the KxY and Pol C motifs. Initial biochemical characterization of piPolBs showed that they share some properties with other replicative DNA polymerases, like a proofreading capacity coupled to 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, structural biology and biochemistry methods to analyze in detail the properties of piPolBs by mutagenesis of clade-specific conserved residues. Namely, the role of an invariable additional aspartate of the Pol A motif and several residues from the piPolBs-specific Pol C motif will be analyzed (see figure).
Ec-373 piPolB was modeled using i- Tasser server, which selected Φ29DNAP (2PYJ) structure as template (A). The Pol C contained loop of modeled piPolB was overlapped with primer-template DNA substrate (B) and Φ29DNAP loop (C).
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.
This project is funded by a Grant entitled Primer-independent DNA polymerases and their applications in biotechnology and biomedicine (ref. PGC2018-093723-A-I00, 2019-2021) from "Generación del Conocimiento" program of Spanish Research Agency.
References:
- Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements (Cell Reports, 2017).
- High diversity and variability of pipolins among a wide range of pathogenic Escherichia coli strains (Scientific Reports 2020).
- Engineered viral DNA polymerase with enhanced DNA amplification capacity. A proof-of-concept of isothermal amplification of damaged DNA (Scientific Reports 2020).