Novel activities and new members of b-family polymerases with applications in biotechnology

Abstract

DNA amplification is a powerful technique widely employed for pathogen detection, biomedical analysis, or genetic research. This process relies on high-fidelity DNA polymerases (DNAPs). One of the mostly used enzymes all over the world is the DNAP from the bacteriophage Φ29 (Φ29DNAP), member of B-family DNAPs (PolBs). This PolB can couple strand displacement capacity with a highly processive and faithful DNA synthesis. These features allowed the development of very efficient isothermal DNA amplification protocols, like multiple displacement DNA amplification (MDA). Conversely, the use of highfidelity DNAPs entails some shortcomings; they are usually blocked by modified bases or DNA lesions, which impairs the amplification of damaged DNA samples. This work aims to increase the variety of DNA amplification methods, providing alternatives for amplification of challenging samples, such as those from forensic or archeological sources as well as metagenomes. Hence, we focused on the study of PolBs with non-canonical activities and the influence of divalent cations on DNA synthesis and amplification proficiency. We show how the DNA amplification yield of the DNAP from bacteriophage Bam35, a processive and faithful PolB with intrinsic strand displacement and translesion DNA synthesis (TLS) capacities, could be greatly enhanced by the fusion of DNA binding domains. Furthermore, we used rolling circle amplification (RCA) assays with synthetic circular singlestranded DNA containing modified bases as template to assess the increment on the processive replication of damaged DNA. Moreover, DNA synthesis opposite to DNA lesions was improved by the employment of Co2+ or a fine-tuned combination of Mg2+ and Mn2+ as cofactors without substantially reducing the enzyme fidelity. In addition, we characterized a new tectiviral PolB infecting a thermoresistant host. This new PolB has proofreading 3’-5’ exonuclease activity, TLS opposite abasic sites and is thermoresistant up to 55 ºC. We have also identified and characterized a new group of PolBs that showed primerindependent DNA synthesis as well as TLS capacity. This new PolB clade, named piPolBs, could be considered a third major group of PolBs, besides protein-primed PolBs and PolBs primed by RNA or DNA. Due to its high processivity and strand displacement capacity, a representative member of the piPolBs group was employed to perform MDA in the absence of primers. Thus, we developed a novel isothermal DNA amplification protocol that only employs one DNAP, without the requirement of exogenous oligonucleotides or primases for priming of the DNA synthesis. Moreover, the yield of this reaction could be greatly enhanced by the addition of the more efficient and processive Φ29DNAP, reaching similar o higher amplification rates than other commercially available methods in the tested conditions. In summary, our results show that some faithful PolBs can combine high processivity and TLS capacity. Thus, the characterization of new naturally occurring DNAPs or engineered enzymes, as well as the variation of metallic cofactors can contribute to the development of methods for isothermal amplification of damaged DNA samples or novel efficient MDA protocols