Single-nucleotide resolution detection of Topo IV cleavage activity in the Escherichia coli genome with Topo-Seq

Abstract

Topoisomerase IV (Topo IV) is the main decatenation enzyme in Escherichia coli; it removes catenation links that are formed during DNA replication. Topo IV binding and cleavage sites were previously identified in the E. coli genome with ChIP-Seq and NorfIP. Here, we used a more sensitive, single-nucleotide resolution Topo-Seq procedure to identify Topo IV cleavage sites (TCSs) genome-wide. We detected thousands of TCSs scattered in the bacterial genome. The determined cleavage motif of Topo IV contained previously known cleavage determinants (−4G/+8C, −2A/+6 T, −1 T/+5A) and additional, not observed previously, positions −7C/+11G and −6C/+10G. TCSs were depleted in the Ter macrodomain except for two exceptionally strong non-canonical cleavage sites located in 33 and 38 bp from the XerC-box of the dif-site. Topo IV cleavage activity was increased in Left and Right macrodomains flanking the Ter macrodomain and was especially high in the 50–60 kb region containing the oriC origin of replication. Topo IV enrichment was also increased downstream of highly active transcription units, indicating that the enzyme is involved in relaxation of transcription-induced positive supercoiling.

Highly sensitive mapping ofin vitrotype II topoisomerase DNA cleavage sites with SHAN-seq

“…8). This periodic nucleo�de bias is consistent with, albeit slightly weaker than, in vivo end-sequencing measurements of gyrase and topo IV cleavage sites and has been associated with the length of DNA gyrase wraps around its CTD 19,20 .…”

“…In addi�on to the effects of quinolones, we found that gyrase and topo IV have dis�nct enzymespecific cleavage site-specifici�es. Although previous in vitro and in vivo studies have found differences in gyrase and topo IV cleavage paterns 16,20 , they have only been compared in the presence of quinolones. Given that quinolones have differen�al effects on gyrase and topo IV 50 , these effects could have accounted for the apparent difference in their cleavage paterns.…”

“…Although we cannot rule out that some individual cleavage sites may s�ll respond to DNA supercoil chirality, our results indicate that it largely serves to independently and globally tune the overall frequency of gyrase and topo IV cleavage complexes on DNA for all cleavage sites. Therefore, supercoiling effects on gyrase and topo IV cleavage complex site-specificity cannot account for why in vivo experiments have found more cleavage complexes on genomic regions that tend to be posi�vely supercoiled [19][20][21] . Instead, these findings may be due to the differen�al binding affinity of the enzymes on nega�vely and posi�vely supercoiled DNA or the compensatory ac�on of type I topos 51 .…”

“…The advent of next-genera�on sequencing technologies has led to new methods for mapping the loca�on and frequency of type II topos on DNA 10,[19][20][21][22][23][24][25] . These methods typically fall into two categories: Chroma�n Immunoprecipita�on sequencing (ChIP-seq), which maps the loca�on of type II topos bound to DNA, and end-sequencing, which maps topo-generated DSBs.…”

“…Lastly, although type II topos recognize DNA supercoiling and supercoil chirality (i.e., nega�ve vs. posi�ve supercoils), these factors are difficult to control and inves�gate in vivo. Recent in vivo studies have found that more type II topo cleavage complexes tend to be associated with posi�vely supercoiled genomic regions than nega�vely supercoiled ones [19][20][21] . However, in in vitro experiments, where DNA supercoiling and supercoil chirality can be precisely controlled, type II topos tend to maintain more overall cleavage complexes on nega�vely supercoiled DNA than on posi�vely supercoiled DNA 26,27 .…”