Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus Under DNA Damage.
Structural maintenance of chromosomes (SMC) proteins play a central role in the organization, segregation and maintenance of chromosomes across domains of life. In bacteria, an SMC-family protein, RecN, has been implicated to have important functions in DNA damage repair. Recent studies have suggested that RecN is required to increase chromosome cohesion in response to DNA damage and may also stimulate specific events during recombination-based repair. While biochemical and genetic assays provide insights into mechanism of action of RecN and other repair factors, in vivo understanding of activity and regulation of proteins can be predominantly gained via microscopy-based approaches. Here, we describe a protocol to study the localization of fluorescently tagged RecN to a site-specific double-strand break (DSB) in Caulobacter crescentus. We further outline a method to probe RecN dynamics in cells with a single, nonreplicating chromosome. This technique can be used to study the early steps of recombination-based repair and understand the regulation of protein recruitment to and further association with sites of damage.
['Chimthanawala|Afroze|A|', 'Badrinarayanan|Anjana|A|']
D001426:Bacterial Proteins D016935:Caulobacter crescentus D020090:Chromosome Segregation D002876:Chromosomes, Bacterial D053903:DNA Breaks, Double-Stranded D004249:DNA Damage D004262:DNA Restriction Enzymes D004269:DNA, Bacterial D005453:Fluorescence D059767:Recombinational DNA Repair
Fluorescence imaging RecN Caulobacter crescentus Bacterial DNA damage Double-strand break repair Microscopy SMC proteins