Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates

Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division

Alternative Sigma Factor σH Modulates Prophage Integration and Excision in Staphylococcus aureus

The prophage is one of the most important components of variable regions in bacterial genomes. Some prophages carry additional genes that may enhance the toxicity and survival ability of their host bacteria. This phenomenon is predominant in Staphylococcus aureus, a very common human pathogen. Bioinformatics analysis of several staphylococcal prophages revealed a highly conserved 40-bp untranslated region upstream of the int gene. A small transcript encoding phage integrase was identified to be initiated from the region, demonstrating that the untranslated region contained a promoter for int. No typical recognition sequence for either σA or σB was identified in the 40-bp region. Experiments both in vitro and in vivo demonstrated that σH recognized the promoter and directed transcription. Genetic deletion of sigH altered the int expression, and subsequently, the excision proportion of prophage DNAs. Phage assays further showed that sigH affected the ability of spontaneous lysis and lysogenization in S. aureus, suggesting that sigH plays a role in stabilizing the lysogenic state. These findings revealed a novel mechanism of prophage integration specifically regulated by a host-source alternative sigma factor. This mechanism suggests a co-evolution strategy of staphylococcal prophages and their host bacteria