JB Accepts, published online ahead of print on 2 June 2014 J. Bacteriol. doi:10.1128/JB.01494-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress

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Justin S. Lenhart1, Eileen R. Brandes1, Jeremy W. Schroeder1, Roderick J. Sorenson2, Hollis D. Showalter2 and Lyle A. Simmons1*

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Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 2

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI

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*Corresponding author Mailing address: 4140 Kraus Natural Science building Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109. Phone: (734) 647-2016. E-mail: [email protected] Fax: (734) 647-0884. Running Title: RecO and RecR load RecA Keywords: RecO, RecR, RecA, DNA replication, recombination

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ABSTRACT RecA is central to maintaining genome integrity in bacterial cells. Despite the near

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ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and

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repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that

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RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following

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DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR

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and RecBCD pathways serve to load RecA and the choice between these two pathways depends

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on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to

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repair centers is strictly dependent on RecO and RecR in response to all types of damage

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examined including a site-specific double-stranded break and damage-independent replication

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fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA

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repair center formation in vivo, RecF does increase the efficiency of repair center assembly,

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suggesting that RecF may influence the initial stages of RecA nucleation or filament extension.

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We further identify single-stranded DNA binding protein (SSB) as an additional component

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important for RecA repair center assembly. Truncation of the SSB C-terminus impairs the ability

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of B. subtilis to form repair centers in response to damage and damage-independent fork arrest.

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With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome

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is necessary for loading and organizing RecA into repair centers in response to DNA damage and

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replication fork arrest.

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INTRODUCTION DNA repair pathways are critical for genome maintenance in all living cells [for review

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(1)]. Both prokaryotic and eukaryotic organisms are constantly exposed to endogenous and

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exogenous sources of DNA damage that compromise genome integrity. In bacterial cells, RecA

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is central to genome integrity and is important for strand exchange during homologous

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recombination, stabilizing stalled replication forks and induction of the SOS transcriptional

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response to DNA damage [for review (2-4)].

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In the Gram-positive bacterium Bacillus subtilis, the process that recruits RecA to excess

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ssDNA has remained unclear (5, 6). In B. subtilis and E. coli, RecA fused to green fluorescent

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protein (GFP) forms foci in response to DNA damage (5-12). The visualization of assembled

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RecA-GFP foci (also referred to as repair centers) provides an in vivo assay to understand the

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events that lead to RecA loading within a living cell.

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In E. coli, RecA is loaded through two major pathways: RecBCD and RecFOR [for

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review (3, 13-15)]. During double-strand break repair, double-stranded ends are processed by the

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RecBCD helicase-nuclease pathway (16). RecBCD generates a 3´ssDNA extension after

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encountering a Chi site (17). Following end processing, RecBCD physically loads RecA onto

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the 3´ssDNA extension (18, 19). The second loading pathway in E. coli is the RecFOR pathway

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(20). In this pathway, RecO and RecR form a complex that is required for RecA loading onto

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SSB coated ssDNA in vitro. The third component, RecF, can accelerate this reaction, particularly

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on gapped DNA substrates (21-23). E. coli, RecA also colocalizes to blocked forks and it was

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shown that RecF and RecO are important for efficient binding of RecA to stalled forks (8, 24).

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Collectively, these studies show that RecA in E. coli is loaded by the RecBCD and RecFOR

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pathways in vitro and in vivo.

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In B. subtilis, the AddAB helicase-nuclease enzyme is required for double-stranded end

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processing and is homologous to E. coli RecBCD (25, 26). Like RecBCD, AddAB generates a 3´

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ssDNA extension suitable for RecA binding and filament formation (25, 26). In contrast to

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RecBCD, B. subtilis AddAB has not been shown to physically bind or directly load RecA. B.

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subtilis also has the RecFOR pathway (27). A notable difference is that B. subtilis RecO is

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necessary and sufficient to load RecA onto SSB-coated ssDNA in vitro (28). Prior work shows

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that RecA-GFP foci were largely recO and recR-independent and defects in AddAB do not

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substantially reduce RecA-GFP foci formation in vivo (5, 29). Therefore, in B. subtilis and other

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AddAB containing bacteria, the process responsible for loading RecA in response to DNA

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breaks, strand gaps, and damage-independent replication fork arrest has remained unknown. This

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is surprising considering that AddAB exists in place of RecBCD in most bacterial organisms

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(30).

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Here we report the proteins that are required for RecA to establish repair centers in

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response to endogenous DNA damage, exogenous sources of DNA damage, and damage-

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independent replication fork arrest. We use RecA-GFP focus formation as a proxy for RecA

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loading in vivo. Importantly, we found that RecA mediator proteins RecO and RecR are

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necessary for RecA to load and organize into repair centers in response to endogenous damage,

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and all sources of exogenous DNA damage and replication fork arrest tested. These results

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support the model that RecOR is critical for RecA filament nucleation on ssDNA in B. subtilis

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and show that AddAB does not direct loading and is dependent on RecOR for RecA loading

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following end processing. We found that RecF, another RecA mediator protein, did not

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influence RecA repair center formation to endogenous damage. However, in response to

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exogenous damage, RecA repair center formation was delayed, in recF deficient cells showing

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that RecF participates in RecA nucleation or enhancement of filament extension. We also found

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that truncation of the C-terminal 35 amino acids of SSB prevents the DNA damage-dependent

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increase in assembly of RecA repair centers, supporting a model where SSB recruits RecA-

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mediator proteins to ssDNA for nucleation and assembly of RecA filaments. With these results,

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we conclude that RecA loading in B. subtilis is dependent on a single loading pathway, providing

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novel insight into the mechanism used to establish RecA repair centers in bacteria with the

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AddAB end processing pathway. RESULTS

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RecA colocalizes to the replisome in response to endogenous and exogenous DNA damage.

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Previous work in E. coli and B. subtilis examined the ability of RecA-GFP to organize into foci

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(5-12, 31). Prior experiments have tested RecA-GFP as the only source of RecA in vivo (6, 8, 9,

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32) GFP-RecA expressed ectopically as the sole source of RecA in cells or in merodiploid cells

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with the native recA allele intact (5, 12). Here, we examined RecA-GFP as the only source of

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intracellular RecA, expressed at its native locus and under control of its native promoter (6). The

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fusion allele was fully functional at low levels of damage (Figure S1 and supplemental results).

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Unless otherwise stated, the experiments described below were performed under conditions of

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DNA damage where the recA-GFP allele was fully functional.

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One current model suggests that RecA predominantly localizes away from the replisome

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in response to DNA damage and that replication is not required for repair center formation (33)

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while other work shows that replication is required for RecA-GFP to form foci (6) and that these

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foci form at midcell (6, 8, 12). To differentiate between these two models, we constructed a

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strain with fluorescent fusions to RecA and the replisome marker DnaX (“Materials and

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Methods”). Imaging of this strain showed that in untreated cells, ~75% of RecA foci colocalized

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with DnaX (replisome) foci. This observation, in consideration with prior work (6), leads us to

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suggest that RecA initiates filament formation to establish repair centers at the replication fork

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followed by a search for homologous DNA. Overall, this result suggests that the bulk of

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endogenous lesions occurring in the growth conditions used here elicit recruitment of RecA to

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the replisome (Figure 1A).

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With this result, we asked if different forms of DNA damage cause RecA to differentially

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localize to or away from the replisome. Our imaging results showed that following challenge

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with mitomycin C (MMC), an agent that forms mostly monoadducts and interstrand crosslinks

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(34), ~65% of RecA foci colocalized with replisomes. Similarly, cells challenged with

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phleomycin, a DNA break inducing peptide (35), showed that ~68% of RecA colocalized with

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replisomes. We found it striking that the majority of RecA foci that assembled in response to

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MMC and phleomycin colocalized with the replisome, supporting earlier work that replication

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fork progression was important for RecA repair center assembly in response to a site-specific

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DSB (6). Following challenge with UV, a treatment that would result in DNA strand gaps and

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replication-stalling thymidine dimers, we found that foci formed immediately (within 5 minutes)

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and ~84% of RecA were colocalized with replisomes (Figure 1). With these results, we conclude

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that the majority of RecA repair centers localized to the replisome in response to endogenous and

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exogenous sources of DNA damage, including phleomycin-generated DNA breaks and MMC-

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generated inter- and intrastrand crosslinks, two types of damage that require homologous

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recombination for repair.

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RecA colocalizes to the replisome in response to damage-independent fork arrest. The

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compound HPUra has been widely used as a tool to rapidly block replication fork progression in

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B. subtilis and other Gram-positive bacteria [e.g. (10, 36-41)]. HPUra is a replication-specific

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class III DNA polymerase inhibitor which blocks replication within minutes (42). To study the

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RecA response to damage-independent replication fork arrest, we chose to use this compound

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because it has been well characterized, but with the limitation that HPUra is not commercially

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available.

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We began by synthesizing HPUra (see Supplemental Information). From prior literature,

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the hydrazine congener H2-HPUra (compound 4) is described as the compound that inhibits

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DNA synthesis [e.g. (36, 39-41)]. We show the scheme for the attempted synthesis of compound

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4 and related congeners in Figure 2A. Briefly, we found that HPUra (compound 3) is readily

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synthesized as described (41). However, numerous attempts to reduce HPUra to H2-HPUra using

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sodium dithionite as described (39), or slight variations thereof, did not provide compound 4 but

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only recovered compound 3. This led us to the conclusion that either compound 3 is active as an

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inhibitor of DNA synthesis or compound 3 is reduced within the cell to produce compound 4. A

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detailed description of the synthesis and structure determination for 3 and congeners is provided

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in Supplemental Information (Figures S2-S12).

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To test compound 3 for DNA synthesis inhibition, we synchronized replication initiation

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using a temperature sensitive initiation mutant as described (6). We analyzed untreated cells 20

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and 60 minutes after replication initiation and determined the position of replication forks by

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quantitative illumina sequencing as described (43). During HPUra treatment, cells were

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challenged with HPUra 20 minutes after replication initiation followed by analysis of DNA

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content at 60 minutes. We show that replication forks in untreated cells progress throughout the

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60 minute time course, while DNA replication in the HPUra treated cells arrests immediately

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upon HPUra addition showing the same DNA content as the 20 minute untreated control. With

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these results, we conclude that, HPUra (compound 3) provides the desired effect of rapidly

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arresting DNA synthesis in vivo (Figure 2B).

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It has been shown that treatment of B. subtilis cells with HPUra blocks DNA synthesis

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and triggers replication stress as measured by RecA-GFP foci formation in most cells (44). We

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found that following HPUra treatment, RecA-GFP formed foci in most cells as rapidly as we

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could prepare the sample for imaging. We found that 76% of cells had RecA-GFP foci at 140

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seconds post treatment (n= 74), and ~94% of cells had RecA-GFP foci 5 minutes after HPUra

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treatment demonstrating the rapid assembly of RecA foci following damage-independent

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replication fork arrest (Figure 1A &C). We performed colocalization with RecA and DnaX and

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found that ~93% of RecA foci (n=181) were colocalized with replisomes following addition of

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HPUra to the growth medium (Figure 1C). These results show that damage-independent fork

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arrest caused RecA to localize to nearly all replisomes in B. subtilis. This work also clarifies the

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method for HPUra synthesis (Supplemental Information).

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RecA-GFP focus formation is dependent on recO and recR. Having established that RecA-

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GFP localized to the replisome in most cells in response to endogenous and exogenous sources

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of damage, we asked which gene product(s) are required for RecA loading, and hence RecA-

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GFP foci formation in vivo. We predicted that recO might contribute to RecA-GFP focus

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formation since B. subtilis RecO helps nucleate formation of RecA onto SSB coated ssDNA in

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vitro (28). To test recO in this capacity, we imaged and quantified RecA-GFP foci in B. subtilis

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cells bearing a recO disruption (recO::cat). In the absence of recO, RecA-GFP foci formed in

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less than 2.1% of cells untreated or following exogenous DNA damage (Figure 3). In addition,

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recO is necessary for damage-independent RecA-GFP filament formation in response to fork

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arrest. This result could be explained by release of the GFP moiety via proteolytic cleavage from

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RecA in a recO mutant. However, we found that RecA-GFP remained intact in the recO::cat

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background (Figure S13A). We also complemented the recO mutant allele via ectopic expression

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(Figure S13B). Together, we found that regardless of the type of damage, RecO is necessary for

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assembly of RecA repair centers in B. subtilis (Figure 3).

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Although B. subtilis RecO and RecR do not form a complex, they do function in the same

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genetic pathway (28, 39). We performed the same experiment in a strain where the recR gene

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was replaced by a cat cassette and obtained nearly identical results to those obtained in recO

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deficient cells (Figure 3). Under all conditions examined, we found that RecA-GFP formed foci

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in 4.5% of cells or less in cells deficient for recR. These results show that recO and recR are both

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necessary for RecA to form foci in response to DNA damage and damage-independent fork

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arrest (please see discussion). This result was not due to proteolytic cleavage of RecA-GFP, and

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this phenotype was complemented by ectopic recR expression (Figure S13B and data not

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shown). We interpret these results to mean that the RecOR pathway is necessary for RecA

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loading in vivo.

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RecF is the third component of the RecFOR pathway, which in E. coli is critical for the

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repair of DNA gap substrates in vivo. This damage is the product of a single-stranded gap in

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otherwise double stranded DNA following UV or mitomycin C exposure. To test the role of

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RecF in RecA-GFP repair center formation, we replaced the recF locus with a cat marker

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(recF::cat) and interestingly, we found that the percentage of cells with RecA-GFP foci was

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mostly unaffected in untreated cells lacking recF (wild type cells untreated: 12.4%+2.6.

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recF::cat untreated: 9.2%+2.1. recO::cat 0.7%+ 0.7 recR::cat strains untreated: 312 for each time point shown.

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Figure 5. SSB is in the recOR pathway. (A) Immunoblot for RecA and DnaN in ssb3+ and

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ssbΔ35 cells. 25 µg of soluble protein extract was applied per lane and separated via SDS PAGE,

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followed by detection with affinity purified RecA antisera in a 1:200 dilution and a 1:2000

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dilution for anti DnaN antiserum. (B) Killing curve of cells with the indicated genetic

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backgrounds over a range of 0 to 0.3 µM MMC. The error bars represent the standard error of

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the mean (SEM).

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Figure 6. Model for RecOR-dependent RecA loading and establishment of repair centers in

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B. subtilis at a DSB. We propose that after AddAB catalyzes end resection to generate a 3´

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ssDNA extension, RecOR is recruited to the large stretches of ssDNA by SSB, and then helps to

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displace SSB and facilitate RecA loading and nucleation of RecA filament formation for

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homology-directed repair.

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Table 1. Truncation of the SSB C-terminal tail eliminates RecA filament formation in response to DNA damage and replication fork stress Relevant Genotype ssb3+ ssbΔ35 ssb3+, recO::cm ssbΔ35, recO::cm ssb3+, recR::cm ssbΔ35, recR::cm

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Exponential Growth 15.0 ± 3.0 20.0 ± 3.1 1.0 ± 1.1 8.8 ± 2.3

40 nM MMC 44.3 ± 2.5 21.5 ± 3.7 0.4 ± 0.7 9.7 ± 3.4

Condition 0.3 μM Phleomycin 46.4 ± 5.4 26.7 ± 4.0 ND ND

0.3 ± 0.6 11.0 ± 3.2

2.8 ± 1.8 11.0 ± 3.1

ND ND

40 μM HPUra

40 J/m2 UV

87.3 ± 3.8 21.6 ± 3.9 0.3 ± 0.6 9.8 ± 2.9

86.5 ± 3.5 22.6 ± 4.3 ND ND

2.9 ± 1.7 13.1 ± 2.8

ND ND

ND indicates “not determined” and error bars represent the 95% confidence interval with n> 282 cells scored for each strain and condition.

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29

A

Colocalization of RecA-GFP with DnaX-mCherry % Colocalization (+/- 95% CI)

Condition

Number of Cells Analyzed

Endogenous (untreated)

74.8 (8.4)

103

40 nM Mitomycin C

65.0 (6.7)

197

84.3 (5.8)

153

400 nM Phleomycin

67.9 (6.3)

212

40 μM HPUra

93.3 (3.3)

223

UV 40 Joules/m

2

Replisome

RecA-GFP

Overlay

B

C

Figure 1.

Phleomycin

Phleomycin

Phleomycin

HPUra

HPUra

HPUra

A

B

0.0

log2(fold enrichment) 0.2 0.4

0.6

60 minutes 20 minutes HPUra

2

Figure 2.

3

0 1 genome position (Mb)

2

Percent of cells with RecA-GFP foci

100 75 50 25 0 WT

Genotype Mitomycin C Phleomycin HPUra UV

Figure 3.

-

+ -

- - -+- -- + -- - +-

recO::cat

+ -

- - +- - +- -+

recR::cat

-

recF::cat

+- - - - +- - - - +- - - -+-

+- - - +- - - +- - -+

A

C

Percentage of cells with RecA-GFP foci

100

80 WT

70 80

60 50

60

40 40

30

recF::cat

20

20

10 0

recO::cat recR::cat

0 0

10

20

30

40

Time (min)

B α-RecA α-SSB recF

Figure 4.

+ -

50

60

0

20

40

60

80

100

120

Time (min) after I-SceI expression

α-DnaN

Relative Survival

α-RecA

bΔ 3

B

1

ss

ss

b3 +

5

A

0.1

0.01

0.001

0.0001

0

0.1 0.2 0.3 Mitomycin C (µM) ssbΔ35

ssb3+

Δ35, recO::cat

ssb3+, recO::cat

Δ35, recR::cat

ssb3+, recR::cat ssb3+, recA::spec

Figure 5.

1) 3´ ssDNA bound by SSB tetramers recruits the RecOR proteins

SSB RecO RecA RecF RecR

2) RecF and RecOR nucleate RecA on ssDNA by displacing SSB

filament growth

3) RecOR facilitate RecA filament growth

Figure 6.