Deaminases and beyond: pathology, physiology and biotechnology.

Genetic abnormalities are the main cause of variability within organisms and are the basis for genetic diseases and cancers. DNA damage caused by AID, APOBEC1 and APOBEC3B was assessed by g- H2AX immunofluorescence, where a strong activation of this marker of DNA damage was observed, thus, inciting DNA disruption and genomic damage. Furthermore, the ability of these cytidine deaminases to induce chromosomal alterations was analysed in 50 individual metaphase spreads by scoring for chromosome aberration. There was an increase in the number of aberrations. Notably, in case of AID, the number of aberration were three times higher than in the control cells, while in the case of APOBEC1 and APOBEC3B, there was a two-fold increase. This clearly indicates the role of AID, APOBEC1 and APOBEC3B causing DNA damage accounting to chromosomal instability. Micronuclei (MN) are an effective cellular indicator of CIN, and elevated frequencies of MN are observed in most solid tumors and pre-neoplastic lesions. In order to investigate the role of the AID/APOBECs as inducers of CIN we are using a quantitative assay for chromosome mis- segregation developed by exploiting the human artificial chromosome (HAC) present in human fibro- sarcoma HT1080 cells. In these cells the HAC kinetochore can be conditionally inactivated, thus leading to formation of micronuclei. Interestingly, we observed elevated levels of formation of micronuclei after transient expression of AID, APOBEC1, APOBEC3A and APOBEC3G in presence of CytochalasinB (CytoB) an actin polymerization inhibitor. Remarkably, even in absence of co- treatment with CytoB, elevated levels of MN were observed especially in the case of AID and APOBEC1. To further verify whether the induction of C to U changes and the downstream activation of the DNA repair pathways, we inhibited Uracil-DNA glycosylase (UDG) using a bacterial UDG inhibitor (UGI). Indeed, a diminution of the levels of MN formation in cells expressing AID and APOBEC1 in the presence of UGI was readily observed, indicating that MN formation is indeed triggered downstream to the activation of the DNA repair pathways. Considering that the AID/APOBECs have been associated to the onset of cancer through their ability to mutate DNA, ourfinding show another possible cancer-inducing effect of these deaminases: their ability to induce chromosomal instability. Recent advances in targeting genomic sequences has revolutionized the genome editing field. Using customized nucleases serves as an ideal platform for targeted deletions, insertion in a broad range of organisms and cell types, but it is less ideal for obtaining changes at specific nucleotides. The fusion of AIDAPOBEC’s to genome editing tool such as Cas9 and TALE has allowed the development of a base editor that aims at avoiding formation of double strand breaks and it offers a new perspective for gene editing. The limit of such base editor is that its targeting is restricted to C•G base pairs. I thus aimed at developing a A•T base editor based on the catalytic domain of ADAR2. Adenosine Deaminases Acting on RNA (ADARs), are editing enzymes that catalyze the C6 deamination of adenosine (A) to produce inosine (I) in double-stranded RNA. A-to- I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. Given the fact that human ADAR2 is able to act on DNA/RNA hybrids, I tried to use chimeras of n/dCas9 and the deaminase domain of ADAR2 to induce a single dA to dG mutation in fluorescent reporter, but our attempts failed. Thus, mutants of ADAR2 with a superior affinity to act on DNA were developed by screening mutagenized libraries through bacterial mutators assays. These mutants may be developed as an alternative base editor to target A:T pairs with potential usage in biotechnological applications such as gene therapy, antiviral treatment and cancer therapy