In Vivo Binding of Recombination Proteins to Non-DSB DNA Lesions and to Replication Forks

Abstract

Homologous recombination (HR) has been extensively studied in response to DNA double-strand breaks (DSBs). In contrast, much less is known about how HR deals with DNA lesions other than DSBs (e.g., at single-stranded DNA) and replication forks, despite the fact that these DNA structures are associated with most spontaneous recombination events. A major handicap for studying the role of HR at non-DSB DNA lesions and replication forks is the difficulty of discriminating whether a recombination protein is associated with the non-DSB lesion per se or rather with a DSB generated during their processing. Here, we describe a method to follow the in vivo binding of recombination proteins to non-DSB DNA lesions and replication forks. This approach is based on the cleavage and subsequent electrophoretic analysis of the target DNA by the recombination protein fused to the micrococcal nuclease.