Cell responses to genome damage

Abstract

The cells' genetic material is constantly under attack, what promotes damage in the DNA molecule. These lesions affect directly DNA replication and RNA transcription, triggering responses that interfere in several metabolic processes, including cell cycle. At the cellular level, unrepaired DNA lesions yield mutations or cell death, which to multicellular organisms result in carcinogenesis and aging. This project intends to study the cell responses triggered after DNA damage, searching to understand the genetic processes implicated in to removal (DNA repair) or in the tolerance to DNA lesions. As these responses are highly conserved, several biological models are employed in these studies, from Prokarya to Eukarya, which give us a broad view on the subject. One of the mostly studied genotoxic agent is ultraviolet (UV) light and part of our aims is to investigate the effects of sunlight (and its UV components) on the DNA molecule. The approach has environmental implications, as it may generate the profiles of DNA lesions, and their biological effects (plasmid inactivation and mutagenesis), induced by the direct exposure to sunlight in different places in earth. The genotoxic effects induced by UV light are also under investigation in human cell lines, proficient and deficient in DNA repair. Specific CPD and 6-4PP-photolyases bearing adenovirus vectors are being used as tools to identify the biological relevance of UVA induced photoproducts, compared to oxidative lesions. We are also investigating cell cycle responses to UVB in synchronized primary human fibroblasts, and the relationship of these responses to the induction of autophagy and cell death, including apoptosis. The effects of these lesions at the level of miRNA and RNA interference metabolism is also under research. Other genotoxic agents, mainly tumor chemotherapeutic agents, will also be investigated concerning the cell mechanisms that promote cell resistance. Mutated cell lines, deficient for different DNA repair pathways are being used for these purposes, together with the development of RNA interference methods (siRNA and shRNA expressed by lentivirus vectors) to promote specific silencing of genes related to DNA lesion processing. These data will not only assist us in the comprehension of cell responses to these agents, as well as they may identify novel gene targets to battle tumor cells. Plant cells' responses to genome damage will also be explored. In this model, we are investigating the expression and the role of classic DNA repair genes, such as xpb orthologs, and genes only recently described as related to DNA lesions responses, such as thi1. We also propose to study specifically the expression and role of sugar cane orthologs for the MutM and Arp genes, linked to base excision repair. In prokaryotes, we plan to conclude functional genomics analysis, with Caulobacter crescentus, from which we identified more than a 100 genes that, when mutated, lead to mutagenesis or to a decrease in cells resistance to genotoxic agents. Our main interest will focus in clones mutated in genes related to DNA metabolism (nucleotide excision repair) and in signal transduction (particularly kinases that participate in the two component systems). These studies will test the correlation of genome lesions and cell cycle, not well known in bacteria. Finally, this project will also address questions related to the dynamic of bacterial genome evolution, using in silico approaches to search for protein signatures (INDELs) in the highly conserved DNA metabolism genes and that evolved through lateral gene transfer. (AU)