Random (UV) Mutagenesis, Unbiased Screening and Identification of DNA Repair Mutants by Complementation Cloning

As a highly radiation-resistant microbial BRCA2 organism, Ustilago maydis is an attractive genetic system to study the mechanisms underlying genome protection. Despite the completion of its genome sequence nearly two decades ago, the functions of many of its 6500+ genes remain unknown. This gap underscores the critical need to identify novel genes, some of which may play vital roles in safeguarding the genome. One effective approach to discover these genes is to conduct forward genetic screens starting with the wild-type and looking for candidates that exhibit pronounced sensitivity to genotoxic agents. Therefore, a united protocol is hereby presented for efficient random mutagenesis, straightforward genetic screening, and the rapid detection of mutated genes by complementation cloning. As the quest for new DNA repair factors continues, it is anticipated that adherence to these procedures will facilitate the discovery of additional factors, thereby shedding light on the molecular networks that coordinate the maintenance of genome integrity in U. maydis and other BRCA2 organisms.