Biology subjects

Goodman, M. F.

Publications and source records attributed to Goodman, M. F..

3 recordsLinked to original sources

bioRxiv2026

Direct visualisation of post-replication gap formation at the bacterial RRS

Replication risk sequences (RRS) are recently discovered genomic structural elements that trigger post-replication gap formation during replisome passage. In E. coli, the two RRS elements are nearly perfect 222-bp G-quadruplex-containing repeats that flank the terminal domain and are highly conserved in both sequence and genomic position across enterobacteria. We report here the first direct visualisation of RRS function in vitro using single-molecule methods. When the G4 strand of the RRS element is positioned on the lagging-strand template, gaps are formed essentially every time a replisome encounters it. An increase in ssDNA in the synthesised DNA is readily seen using ssGAP-seq methods. When the G-quadruplex strand of the RRS is positioned on the leading-strand template, gaps are formed, albeit at lower frequency. However, the continued DNA synthesis in a rolling-circle assay indicates that the gaps are still formed on the lagging strand, indicating that the RRS complementary strand has a significant but reduced capacity to form a structure that triggers lesion skipping. The results document the potency of the RRS as a trigger for gap formation, suggesting a possible function for at least some eukaryotic G-quadruplexes.

biophysics↗

bioRxiv2023

Controlling Genome Topology with Sequences that Trigger Post-replication Gap Formation During Replisome Passage: The E. coli RRS Elements

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, [≤]2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

genomics↗

bioRxiv2019

UmuD and RecA* modulate the DNA-binding activity of DNA polymerase IV in Escherichia coli

DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells suffering high levels of DNA damage. In a recent single-molecule imaging study, we demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD, which accumulates in DNA-damaged cells, inhibits binding. In agreement with proposed roles for pol IV in homologous recombination, up to 40% of pol IV foci colocalise with a probe for RecA* nucleoprotein filaments in ciprofloxacin-treated cells. A hyperactive RecA mutant, recA(E38K), allows pol IV to bind the nucleoid even in the absence of exogenous DNA damage. In vitro, RecA(E38K) forms RecA*-like structures that can recruit pol IV, even on double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results indicate that UmuD and RecA modulate the binding of pol IV to its DNA substrates, which frequently coincide with RecA* structures.

microbiology↗