Processing of stalled replication forks in Bacillus subtilis

Abstract Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis, which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography, and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, and RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, and PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, and PriA), Holliday junction resolvase (RecU), nucleases (RnhC and DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are key functions required to overcome a replication stress, provided that the fork does not collapse.


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RecG unwinds stalled forks to reverse them, and regresses a HJ DNA leading to fork restoration (Cañas 381 et al., 2014, Torres et al., 2021).These fork remodeling activities mediated by RecG are believed to 382 contribute to PriA-dependent replication restart.

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RecD2 is a 5'3' helicase (Walsh et al., 2014) with branch migration activity on three-strand 384 recombination intermediates (D-loops) (Ramos et al., 2022), but its activity on the remodeling of stalled 385 or reversed forks remains elusive.RadA/Sms, which is a ring shaped hexameric 5´3´ DNA helicase, unwinds DNA in the presence of an available 5´-tail without the need for any accessory protein (Marie 387 et al., 2017, Torres et al., 2019a).Acting as a mediator, RadA/Sms partially displaces SsbA and facilitates RecA nucleation on the 5´-fork DNA (Torres et al., 2019c, Hertzog et al., 2023, Torres et al., 389 2023).Subsequently, RecA acting as a loader, activates RadA/Sms to unwind several structures, such as 390 mobile D-loops, 5´-fork DNAs, or reversed forks with a nascent leading-strand longer than the nascent 391 lagging-strand (Torres et al., 2019a, Torres & Alonso, 2021, Torres et al., 2023).The fork clearance 392 activity mediated by RadA/Sms is believed to be important to create the proper substrate for PriA-393 dependent replication restart.(Witte et al., 2008, Gándara & Alonso, 2015).In response to DNA damage, DisA  et al., 2007, Giramma et al., 2021, Kruger et al., 2021).contained spontaneous static foci (Gándara et al., 2017).Exponentially growing cells also contain a 441 similar number of spontaneous RecA foci and the large majority of these foci colocalize with stalled 442 forks (Simmons et al., 2007, Lenhart et al., 2014).In vitro studies revealed that DisA interacts with and 443 inhibits RecA-mediated DNA strand exchange (Torres et al., 2019c).This result suggests that at least 444 some repair mechanisms to reactivate stalled forks may not require the strand exchange activity of 445 RecA.

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Live cell studies have revealed that RadA/Sms also forms dynamic foci that colocalize with the 447 nucleoid in 63% of the cells, but DisA and RadA/Sms foci only transiently colocalize (in ∼27% of 448 cells) (Gándara et al., 2017).In the recG and recU mutants, in which branched intermediates (stalled or reversed forks) accumulate in vivo (Carrasco et al., 2004, Sanchez et al., 2007) for DNA repair (Gándara & Alonso, 2015, Gándara et al., 2017).The physical interaction among the proteins that show a genetic and biochemical interplay upon 467 replication stress has been analyzed using pull-downs or a bacterial two-hybrid system in vivo and some 468 of them have been confirmed through different in vitro protein-protein interaction assays (Figure 2).

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These analyses show a dense interconnexion between many proteins, highlighting the importance of an indirect cross-talk with the RecA and SsbA hubs.Similarly, RecU is indirectly part of the RecA and (Figure 2) (Carrasco et al., 2005, Cañas et al., 2008, Carrasco et al., 2009, Cañas et al., 2011).

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Remarkably, RnhC interacts with RNAP even in the absence of exogenous DNA damage, highlighting 492 the importance of the resolution of RTCs (Delumeau et al., 2011).Furthermore, certain functions of the 493 translation complex interact with DinG (Costes et al., 2010).Among these protein interactions, it can be 494 highlighted that PcrA and RnhC are part of RecA and RNAP protein-interaction hubs (Figure 2).

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To avoid replication fork collapse and ultimately maintain genome stability, stalling impediments 519 must be repaired, circumvented or bypassed efficiently before replisome reloading.RecA-GFP, 520 expressed from its native locus and under the control of its native promoter, is largely cytosolic in 521 unstressed cells (Simmons et al., 2007).However, several reports have shown that 15% of total that is subsequently reengaged upon DnaG-mediated repriming (Figure 1A) (Henry et al., 2023)  are shared with E. coli (Au et al., 2005).

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This apparent paradox might be explained by the existence of two distinct Rad51 activities: canonical The non-recombinogenic or non-canonical RecA activities refer to those activities that are they are difficult to prove in live cells.However, based on available information, we propose that in the 673 absence of ATP hydrolysis, RecA may protect the fork from degradation, as it has been observed for 674 Rad51 (Zellweger et al., 2015), and help to recruit other proteins for fork processing.There are few 675 pieces of information that indirectly suggest a non-canonical activity of RecA in B. subtilis.First, RecA 676 forms foci that are not converted into threads at RTCs or in response to a low UV dose (1 J/m 2 ) 677 (Simmons et al., 2007, Million-Weaver et al., 2015) (Simmons et al., 2007).Non-canonical RecA activities may protect stalled forks from degradation, and 691 may contribute to suppressing the uncoupling of ongoing replication forks to limit ssDNA accumulation 692 at stalled forks.should be a minor pathway (Figure 1 D-E), because it could be mutagenic (Aliotta et al., 1996, Sung et 704 al., 2003, Duigou et al., 2004, Duigou et al., 2005) difficult to obtain, is not available.Thus, the present evidence of this process relies on in vitro branch migrate a fork reversed by RecG (Gándara et al., 2021).RuvAB-mediated branch migration may components of the replisome to enable replication restart (Haroniti et al., 2003, Bailey et al., 2007, positive supercoils built up at HO RTCs (Lang & Merrikh, 2021).Furthermore, DNA gyrase was found 892 to drive pervasive R-loop formation, that should require replication restart (Lang & Merrikh, 2021).

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Finally, Topo I depletion rescues rrnIHG-inverted cells from a severe growth defect when grown in LB 894 medium (Yeesin, 2019), although the molecular mechanism remains elusive.Whether Topo III is 895 required to resolve RTCs in B. subtilis remains to be tested.
remodeling (Soultanas et al., 2000, Polard et al., 2002, Qin et al., 2014, Matthews & stress sensed in B. subtilis cells?The DisA checkpoint protein was originally 398 described as a safeguard of genome integrity in B. subtilis sporulating cells.DisA scans the 399 chromosome, and delays entry into sporulation in the presence of DNA damage (Bejerano-Sagie et al., 400 2006).Single-molecule fluorescent imaging cell analysis revealed that in a large majority of unstressed 401 sporulating cells (88%), DisA forms a highly dynamic focus that transiently associates with and 402 dissociates from the nucleoid, moving rapidly along the chromosome scanning for "perturbations" 403 (Bejerano-Sagie et al., 2006, Torres et al., 2019c).Transient binding to DNA is required for DisA 404 scanning and pausing, since its variant lacking the RuvA-like DNA binding domain (DisA∆C290) 405 forms a focus that moves freely on the cytosol (Torres et al., 2019c).406 While scanning, DisA synthesizes the essential second messenger cyclic 3', 5'-diadenosine 407 monophosphate (c-di-AMP) (Oppenheimer-Shaanan et al., 2011), being the major c-di-AMP synthase 408 in B. subtilis cells

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522unstressed cells contain RecA foci that colocalize with the nucleoid, and the large majority of these foci 523 Downloaded from https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuad065/7459347 by guest on 08 December 2023 midcell or at quarter-cell positions, as the replisome (>85% colocalization with DnaX) Eco and with DnaG Eco , colocalizes with the replisome(Henrikus et al., 2019, Henry   561   et al., 2023).It has been proposed that the RecF Eco -replisome interaction may destabilize the replisome, 562 reviewed within the previous sections suggests that replication stress in B. subtilis, 697 often causes replisome disassembly, and that the repair, modulated by RecA, is spatially and 698 temporarily coupled with DNA replication.Consequently, lesion skipping and post-replicational repair 699 of the gap left behind (Figure 1A-C) are likely not the primary mechanisms employed to overcome 700 transient stalled replisomes in B. subtilis.This coupling between replication and repair would limit 701 ssDNA formation, and indirectly protect the stalled or reversed fork.The nature of the replication stress 702 is a key determinant of the chosen pathway.PolC holoenzyme replacement by either PolY1 or PolY2, 703

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which is an important source of genome instability, is an inherent challenge that 923 DNA replication processes face due to the various obstacles encountered by the replisome, including 924 stalled transcription machineries, bound proteins, and endogenous DNA damage.B. subtilis utilizes 925 multiple mechanisms to coordinate rescue of stalled replication forks, and failure in this repair results in 926 defects in transcription elongation, DNA damage and chromosomal segregation (Anderson et al., 2022).927 Cells have evolved a repertoire of strategies to handle replication stress, and the choice among these 928 Downloaded from https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuad065/7459347 by guest on 08 December 2023 depending on the specific context and possible outcomes.Therefore, the multiple 929 forms of differentiation and development of the B. subtilis bacterium may help us to define the proteins 930 involved in different situations.Genetic works carried out over 50 years have contributed to our 931 comprehension of the functions that contribute to cope with replication stress.Many proteins still 932 remain to be analyzed, and our understanding of the DNA damage-dependent but SOS-independent 933 regulation is yet poor due to the intricate interplay of multiple regulators.934 In the past two decades, single-molecule fluorescence observations, genomic and proteomic 935 analyses in live bacteria, and biochemical studies have helped us to the reconstruction of mechanisms 936 employed by cells to manage replication stress and RTCs.These investigations are unraveling the 937 pathways chosen by cells, and the roles of the various proteins involved in these intricate processes.It is 938 conceivable that future studies will benefit from methodologies aimed at determining helicase loading 939 and dynamics in response to diverse types of replication stress, and the contribution of error-prone TLS 940 polymerases, that is still poorly understood in B. subtilis cells.941 Another critical area of future investigation is the comprehension of the biological contribution of 942 non-canonical RecA activities to the choice of the pathway to alleviate replication stress, and the 943 coordination of the checkpoints and fork remodelers.The identification and study of mutants in which 944 different RecA activities are specifically inactivated should help us to understand non-canonical RecA 945 mechanisms.Numerous protein-protein interactions among the different functions that mitigate 946 replication stress have been documented, but structural information of assembled complexes is still 947 needed to fully understand these interactions and how they are coordinated.It is also necessary to 948 unravel the contribution in response to replication stress of secondary metabolites ([p]ppGpp, c-di-949 AMP, and potentially unidentified compounds, such as the one synthesized by CczA (Wozniak et al., 950 2022)), as well as the effect of levels of replication proteins and dNTPs imbalance.Furthermore, 951 understanding the loading of primosomal proteins (DnaC, DnaG and DnaE) during replication restart, 952 and their particular coordination on hybrid primer synthesis on the nascent lagging-strand, requires 953 further studies.954 B. subtilis serves as a valuable model for deciphering the molecular mechanisms and the crucial 955 proteins involved in overcoming replication stress via error-free DDT in pathogenic bacteria of the 956 Bacilli Class (Staphylococcus, Streptococcus, Enterococcus, etc.).A better overview of these processes 957 could help us to develop novel targets for the development of safe and effective antimicrobial agents.López-Sanz, Chiara Marchisone and Paula Pérez Junquera for technical assistance of 961 work in our laboratory over the years.962963 Downloaded from https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuad065/7459347 by guest on 08 December 2023 supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/ 966 10.13039/501100011033/ FEDER, EU [PID2021-122273NB-I00] and intramural [CSIC 2021AEP031] Chatelier E, Green M, Nouri H, Kepes F, Soultanas P & Janniere L (2017) Interactions 1301 of the Bacillus subtilis DnaE polymerase with replisomal proteins modulate its activity and fidelity.1302 Open Biol 7. 1303 Pei HH, Hilal T, Chen ZA, et al. (2020) The  subunit and NTPase HelD institute a two-pronged 1304 mechanism for RNA polymerase recycling.Nat Commun 11: 6418.Kidane D, Reed P, Curtis FA, Cozar MC, Graumann PL, Sharples GJ & Alonso JC (2005) 1342 The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for 1343 double-stranded DNA break repair in Bacillus subtilis.Genetics 171: 873-883.1344 Sanders GM, Dallmann HG & McHenry CS (2010) Reconstitution of the B. subtilis replisome with 13 1345 proteins including two distinct replicases.Mol Cell 37: 273-281.
In vitro DisA does not affect PriA- (Bejerano-Sagie et al., 2006)art, but it slightly increases the length of the Okazaki fragments(Raguse et   415   al., 2017), an effect that has been also observed when DnaG concentrations are lowered(Seco et al.,   4162013).It is likely that DisA plays a fail-safe mechanism to ensure complete and accurate DNA 417 replication before the cell enters in the sporulation state(Bejerano-Sagie et al., 2006).418Tounderstand which signal(s) are being recognized by and pause DisA movement upon DNA 419 damage, both in vivo and in vitro experiments were conducted.It was observed that in vitro DisA , DisA-YFP formsIn vitro, DisA limits the activity of many proteins that act at the stalled fork.DisA bound to stalled 454 2021, Torres et al., 2023).However, DisA bound to a branched intermediate neither affects PriA-455 dependent replication re-start (Raguse et al., 2017) nor PcrA-mediated DNA unwinding (Torres et al., 456 2021), suggesting that the inhibitory activity of DisA over some recombination proteins is protein 457 specific.458 All these analyses suggest that upon replication stress branched intermediates accumulate, 459 inducing DisA pausing, blocking c-di-AMP synthesis, and thereby indirectly inhibiting DnaG (Wang et 460 al., 2007, Gándara et al., 2017).Consequently, DisA pausing might decrease the overall velocity of the 461 sister replisome, perhaps to coordinate the clockwise and counterclockwise replisomes and allow time 462 . All 563 these results suggest that in E. coli the PolIII Eco holoenzyme usually skips over the lesion to leave May these recombination B. subtilis proteins alter replisome dynamics?In vitro two set of 569 activities performed by recombination proteins during replication re-initiation have been observed: 570 those that directly inhibit replication re-initiation, and those that indirectly impair replication 571 elongation.Within the first activity group, RecA is included.In vitro, RecA, with the help of RecO and (Elsholz et al., 2012, Schmidt et al., 2014)ion re-initiation from a DNA substrate that mimics a 3´-fork DNA 573(Vlasic et al., 2014).RecD2, which promotes RecA disassembly from ssDNA, plays a dual role in vitro: 574 it antagonizes the negative effect exerted by RecA on PriA-dependent DNA replication restart, but at 575 high concentrations inhibits DNA replication restart(Ramos et al., 2022).RarA, at a 3´-fork DNA 576 substrate, also inhibits PriA-dependent replication initiation(Carrasco et al., 2018).Notably, all these 577 effects are at the restart step, because in vitro DNA replication elongation remains unaffected by RecA, 578 RecO, RarA, or RecD2(Vlasic et al., 2014, Carrasco et al., 2018, Ramos et al., 2022).The second 579 activity is performed by DisA, and it is related to the fact that in vivo, low levels of c-di-AMP, upon 580 DisA binding to a branched intermediate, indirectly inhibit the DnaG activity (see above) (Wang et al., 581 2007).588subtilis, a fraction of RecA and SsbA is phosphorylated(Elsholz et al., 2012, Schmidt et al., 2014).589 However, the physiological role of protein phosphorylation in response to replication stress remains 593 In B. subtilis, the application of different impediments to replication fork progression has been 594 used to analyze the responses to a replication stress.First, transient arrest of only one replisome (for 90 595 min), by repressors binding to a discrete operator array located specifically at one arm of the replisome, 596 leaving replication of the other arm and replication re-initiation unaffected, was assayed.Under this (Kidane et al., 2009e of cell, Walsh et al., 2014)threads significantly increased(Lenhart   637   et al., 2014, Walsh et al., 2014).RecA threads are short-lived in recF15 or rarA cells, but long-lived 638 in recD2, recX, and recU cells(Kidane et al., 2009, Cárdenas et al., 2012, Carrasco et al., 2018, (Torres et al., 2019a, Torres et al., 2019c, Carrasco et al., 2022isA or RadA/Sms, which inhibit the ATPase and the DNA strand exchange 679 activities of RecA, perhaps to prevent RecA from engaging in unnecessary homology search and strand 680 exchange(Torres et al., 2019a, Torres et al., 2019c, Carrasco et al., 2022, Carrasco et al., 2023)Third, (Gomez-Gomez et al., 2007)uang et al., 2023) 2010, Huang et al., 2023), PcrA overexpression improves 683 viability of recA + cells, but not of recA cells.Contrarily, upon rrnIHG inversion, overexpression of 684 RnhC does not improve the viability of recA + cells grown on LB, but slightly increases viability of 685 recA cells(Yeesin, 2019).Thus, it is likely that a non-canonical RecA activity cooperates with PcrA 686 to overcome HO RTCs, and with RnhC to remove R-loops.Finally, RecA promotes swarming motility, 687 and such effect does not require canonical RecA activities(Gomez-Gomez et al., 2007).688Whetherthese non-canonical activities can be also performed by other bacterial RecA homologs is 689 unknown.In B. subtilis, RecA foci formation at locations distal from replisomes is rarely observed 690 Downloaded from https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuad065/7459347 by guest on 08 December 2023 . It is tempting to speculate that the non-canonical 705 activity of RecA (see above) licenses fork remodeling, serving as the primary avenue to cope with 706 replication stress.Direct in vivo documentation of how fork remodeling occurs, which has proven 707 is 901 required to ameliorate HO RTCs.Indeed, PcrA over-expression increases plating efficiency of rrnIHG-902 inverted cells grown in LB, but such effect is not observed in the absence of RecA.Contrarely, RnhC 903 over-expression does not significantly affect the plating efficiency, both in the presence or the absence Alleviation of RTCs in E. coli has been shown to occur differently.When the replisome collides 906 with RNAP Eco on the template leading-strand, both machineries transiently pause.The Rep Eco and 907 UvrD Eco translocases are recruited to the stalled fork, with Rep Eco interacting with DnaB Eco , and UvrD Eco 908 905 al., 911 2019, Whinn et al., 2023).When the E. coli replisome encounters a cluster of RNAPs on the template 912 lagging-strand due to the inversion of the rrnA Eco or the rrnE Eco and rrnB Eco operons, both machineries 913 stall, leading to a HO RTC that does not significantly affect colony formation in wt, recA Eco or , De 916 Septenville et al., 2012).The specific enzyme(s) that reverses the stalled forks remains unidentified (De 917 Septenville et al., 2012).Remarkably, the resolution of RTCs at HO sites requires RecBCD Eco , or 918 DinG Eco , but not RecA Eco