R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided.
- Dirk Remus
- Dirk Remus
- Dirk Remus
- Jack D Griffith
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bruce Stillman, Cold Spring Harbor Laboratory, United States
© 2021, Kumar et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.
Six transmembrane epithelial antigen of the prostate (STEAP) 1–4 are membrane-embedded hemoproteins that chelate a heme prosthetic group in a transmembrane domain (TMD). STEAP2–4, but not STEAP1, have an intracellular oxidoreductase domain (OxRD) and can mediate cross-membrane electron transfer from NADPH via FAD and heme. However, it is unknown whether STEAP1 can establish a physiologically relevant electron transfer chain. Here, we show that STEAP1 can be reduced by reduced FAD or soluble cytochrome b5 reductase that serves as a surrogate OxRD, providing the first evidence that STEAP1 can support a cross-membrane electron transfer chain. It is not clear whether FAD, which relays electrons from NADPH in OxRD to heme in TMD, remains constantly bound to the STEAPs. We found that FAD reduced by STEAP2 can be utilized by STEAP1, suggesting that FAD is diffusible rather than staying bound to STEAP2. We determined the structure of human STEAP2 in complex with NADP+ and FAD to an overall resolution of 3.2 Å by cryo-electron microscopy and found that the two cofactors bind STEAP2 similarly as in STEAP4, suggesting that a diffusible FAD is a general feature of the electron transfer mechanism in the STEAPs. We also demonstrated that STEAP2 reduces ferric nitrilotriacetic acid (Fe3+-NTA) significantly slower than STEAP1 and proposed that the slower reduction is due to the poor Fe3+-NTA binding to the highly flexible extracellular region in STEAP2. These results establish a solid foundation for understanding the function and mechanisms of the STEAPs.