The Rice miR396-GRF-GIF-SWI/SNF Module: A Player in GA Signaling

Rice Growth-Regulating Factors (GRFs) were originally identified to be gibberellin (GA)-induced, but the nature of GA induction has remained unknown because most reports thereafter focused on revealing their roles in growth-promoting activities. GRFs have the WRC (Trp, Arg, Cys) domain to target DNA and contain the QLQ (Gln, Leu, Gln) domain to interact with GRF-Interacting Factor (GIF), which recruits ATP-dependent DNA translocase Switch/Sucrose Non-fermenting (SWI/SNF) for chromatin remodeling. Both GRFs and GIFs exhibit transcriptional activities but GIFs lack a DNA-binding domain. So, GRFs act like a navigator in the GRF-GIF-SWI/SNF complex, determining when and where the complex should work on. The levels of most rice GRFs can be sensitively regulated by miR396, which responds to many developmental and environmental factors. Recent clues from several studies highlight the original question of how GRFs participate in GA signaling. DELLA (contain DELLA motif) protein plays dual roles in controlling the level of GRFs by regulating the level of miR396 and interacting with GRFs. Here we address the question of why this complex plays an essential role in controlling plant growth focusing on the action of GA signaling pivot, DELLA.

Natural transformation protein ComFA exhibits single-stranded DNA translocase activity

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a handful of proteins required for natural transformation in gram-positive bacteria. Its structural resemblance to the DEAD-box helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5′ to 3′. However, this translocase activity does not lead to DNA unwinding in the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake.

Membrane fission during bacterial spore development requires DNA-driven cellular inflation

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions.

RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

Mfd regulates RNA polymerase association with hard-to-transcribe regions in vivo, especially those with structured RNAs

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement and influence transcription, ultimately necessitating the activity of RNAP-associated factors. One such factor is the bacterial protein Mfd, a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfd’s in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd’s impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard to transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism to transfer RNA regulation to toxin–antitoxin (TA) functions. We found that cells lacking Mfd are highly sensitive to toxin overexpression. Finally, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP cofactor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.

Mfd regulates RNA polymerase association with hard-to-transcribe regions in vivo, especially those with structured RNAs

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement, influence transcription, ultimately necessitating the activity of RNAP associated factors. One such factor is the bacterial protein Mfd; a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized.To shed light on Mfd’s in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd’s impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard-to-transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism, to tRNA regulation, to toxin-antitoxin (TA) functions. We found that transcription regulation by Mfd, at least at some TA loci, is critical for cell survival. Lastly, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems, and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP co-factor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.SignificanceThe bacterial DNA translocase Mfd binds to stalled RNAPs and is generally thought to facilitate transcription-coupled DNA repair. Most of our knowledge about Mfd is based on data from biochemical studies. However, little is known about Mfd’s function in living cells, especially in the absence of exogenous DNA damage. Here, we show that Mfd modulates RNAP association and alters transcription at a variety of chromosomal loci, especially those containing highly structured, regulatory RNAs. As such, this work improves our understanding of Mfd’s function in living cells, and assigns it a new function as a transcription regulator.

A DNA translocase operates by cycling between planar and lock-washer structures

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport substrates possessing a preexisting helical structure, such as the bacteriophage ϕ29 dsDNA packaging motor. During each cycle, this pentameric motor tracks one helix strand (the ‘tracking strand’), and alternates between two segregated phases: a dwell in which it exchanges ADP for ATP and a burst in which it packages a full turn of DNA in four steps. We challenge this motor with DNA-RNA hybrids and dsRNA substrates and find that it adapts the size of its burst to the corresponding shorter helical pitches by keeping three of its power strokes invariant while shortening the fourth. Intermittently, the motor loses grip when the tracking strand is RNA, indicating that it makes load-bearing contacts with the substrate that are optimal with dsDNA. The motor possesses weaker grip when ADP-bound at the end of the burst. To rationalize all these observations, we propose a helical inchworm translocation mechanism in which the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. However, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

PICH function is required for organization of SUMOylated proteins on mitotic chromosomes

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by abnormal distribution of genetic information. Polo-like kinase 1 interacting checkpoint helicase (PICH) is a DNA translocase essential in chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by Small Ubiquitin-like modifier (SUMO). However, it is unclear how these activities are cooperating to resolve chromosome bridges. Here, we show that PICH specifically promotes the organization of SUMOylated proteins like SUMOylated TopoisomeraseIIα (TopoIIα) on mitotic chromosomes. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in the retention of SUMOylated chromosomal proteins, including TopoIIα, indicating that PICH functions to control proper association of these proteins with chromosomes. Replacement of PICH with its mutants showed that PICH is required for the proper organization of SUMOylated proteins on chromosomes. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taken together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.Summary StatementPolo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH controls association of SUMOylated chromosomal proteins, including Topoisomerase IIα, and that function requires PICH translocase activity and SUMO binding ability.

PICH promotes SUMOylated TopoisomeraseIIα dissociation from mitotic centromeres for proper chromosome segregation

Polo-like kinase interacting checkpoint helicase (PICH) is a SNF2 family DNA translocase and is a Small Ubiquitin-like modifier (SUMO) binding protein. Despite that both translocase activity and SUMO-binding activity are required for proper chromosome segregation, how these two activities function to mediate chromosome segregation remains unknown. Here, we show that PICH specifically promotes dissociation of SUMOylated TopoisomeraseIIα (TopoIIα) from mitotic chromosomes. When TopoIIα is stalled by treatment of cells with a potent TopoII inhibitor, ICRF-193, TopoIIα becomes SUMOylated, and this promotes its interaction with PICH. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in retention of SUMOylated TopoIIα on chromosomes, indicating that PICH removes stalled SUMOylated TopoIIα from chromosomes. In vitro assays showed that PICH specifically regulates SUMOylated TopoIIα activity using its SUMO-binding and translocase activities. Taken together, we propose a novel mechanism for how PICH acts on stalled SUMOylated TopoIIα for proper chromosome segregation.Summary StatementPolo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH promotes dissociation of SUMOylated TopoisomeraseIIα from chromosomes and that function leads to proper chromosome segregation.