Molecular and functional characterization of the Listeria monocytogenes RecA protein: Insights into the homologous recombination process

Journal Pre-proof Molecular and functional characterization of the Listeria monocytogenes RecA protein: insights into the homologous recombination process Debika Ojha, K. Neelakanteshwar Patil

PII:

S1357-2725(19)30219-5

DOI:

https://doi.org/10.1016/j.biocel.2019.105642

Reference:

BC 105642

To appear in:

International Journal of Biochemistry and Cell Biology

Received Date:

11 May 2019

Revised Date:

20 October 2019

Accepted Date:

31 October 2019

Please cite this article as: Ojha D, Neelakanteshwar Patil K, Molecular and functional characterization of the Listeria monocytogenes RecA protein: insights into the homologous recombination process, International Journal of Biochemistry and Cell Biology (2019), doi: https://doi.org/10.1016/j.biocel.2019.105642

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Molecular and functional characterization of the Listeria monocytogenes RecA protein: insights into the homologous recombination process

Debika Ojha 1, 2 and K. Neelakanteshwar Patil 1, 2 * From the 1 Department of Protein Chemistry and Technology, Council of Scientific & Industrial Research-Central Food Technological Research Institute (CSIR-CFTRI),

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(AcSIR), Ghaziabad 201002, Uttar Pradesh, India.

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Mysuru 570 020, Karnataka, India, 2 Academy of Scientific and Innovative Research

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*To whom correspondence should be addressed: K Neelakanteshwar Patil, Department of Protein Chemistry and Technology, Council of Scientific and Industrial

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Research-Central Food Technological Research Institute, Mysuru 570 020,

https://orcid.org/0000-0002-9402-1341

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ORCID ID:

e-mail: [email protected]

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Karnataka, India,

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Graphical abstract

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A Nucleoprotein + dsDNA filament

6-FAM labeled + RecA ssDNA

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Time (min) 2 5 10

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-RecA

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LmRecA 0.25 0.5

Displaced Hetero duplex + ssDNA

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0.25 0.5

LmRecA K70A Time (min) 1 2 5 10

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Hetero duplex

ssDNA 1

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Highlights

L. monocytogenes RecA gets expressed more than 2 fold in response to DNA

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damaging agents MMS and UV. The purified LmRecA protein catalyses D-loop formation, strand exchange

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activity and hydrolyses ATP.

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The mutant LmRecAK70A protein shown reduced strand exchange activity and failed to hydrolyze ATP, showing the conserved walker motifs.

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The LmRecA cleaves LexA protein and protects RecA presynaptic filament from Exonuclease I activity.

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This is the first report on biochemical characterization of L. monocytogenes RecA protein.

Abstract The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and

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transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the

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DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The

purified L. monocytogenes RecA protein show robust binding to single stranded

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DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP,

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whereas the mutant LmRecAK70A fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecAK70A perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an

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oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from

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the exonuclease I activity. Altogether, this study provides the first detailed

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characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.

Keywords: Recombination; RecA; RecA nucleoprotein filament, SOS response; Listeria monocytogenes, ATPase. 3

Introduction Homologous recombination (HR) is conserved across all organisms. It plays an important role in cellular homeostasis by maintaining genome integrity through the repair of double-strand breaks (DSB) as well as genetic diversification through meiotic recombination [1–3]. One of the key steps in the HR process, is DNA strand

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exchange catalyzed by the RecA family of proteins which are ubiquitous and wellconserved among bacterial species [4–7]. The RecA protein binds and hydrolyzes ATP; this feature enables it to bind DNA in a sequence non-specific manner and

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promote DNA strand exchange [5, 8]. Kinetically, the HR process is divided into

three phases: (i) presynapsis, (ii) pairing and strand exchange and (iii) resolution [9–

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11]. In this reaction, RecA protein first binds to the single stranded DNA (ssDNA) in the presence of ATP to form the RecA nucleoprotein or presynaptic filament [12–14].

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During synapsis, the RecA nucleoprotein filament binds to double stranded DNA (dsDNA), locates the site of homology and catalyzes the exchange of strands to

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produce the Holliday junction intermediate [15–16]. Finally, the RuvABC complex resolves the resolution by processing the recombinant intermediates [9]. RecA also

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plays a key role in SOS induction via coproteolytic cleavage of the LexA repressor

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and to induce the expression of 40 genes involves in this pathway [17–20]. Listeria monocytogenes is ubiquitous in the environment and is a gram-positive

foodborne pathogen; it is a causative agent of listeriosis and is associated with meningitis, encephalitis and spontaneous abortions [21]. The threat of listeriosis is a significant public health risk because of the high fatality rate, ranging from 15 to 30 deaths per 100 cases [21]. L. monocytogenes is a highly-contagious pathogen and is

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transmitted through contaminated food, mainly meat, dairy products and poultry as well as ready-to-eat pre-packaged products [22]. This pathogen can adapt and grow in extreme environmental conditions like high osmolarity, low pH and low temperature during food processing as well as acid and bile gastrointestinal stress during host infection [23]. The threat of L. monocytogenes is severe as it forms biofilms that are known to resist antimicrobials. In France, the first multidrug-resistant L. monocytogenes was isolated in 1988 from a patient suffering from

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meningoencephalitis [24]. Since then, many antibiotic-resistant L. monocytogenes strains have been isolated from the environment, food, infected humans and animals [25]. In this context, recent studies have shown that by actively participating in

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stress-induced DNA repair, RecA helps in conferring antibiotic resistance to

microorganisms [26–27 and references therein]. Therefore, it is necessary to

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understand the biochemical function of the RecA protein in L. monocytogenes.

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In the present investigation, we have characterized the RecA protein from L. monocytogenes. Results show that, L. monocytogenes RecA (LmRecA) protein is

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elevated in response to DNA damaging agents. The purified protein binds to both ATP and ssDNA and forms active nucleoprotein filaments that catalyse displacement

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loop (D-loop) formation, three-strand exchange reaction and cleaves the repressor LexA. The RecA protein also inhibit 3’ exonuclease I (Exo I) activity to protects the

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DNA from degradation. The LmRecAK70A fails to hydrolyze ATP, showing conserved ATPase walker A and B motifs. These results shed light on the canonical activities of the RecA protein from a foodborne pathogen. Altogether, this is the first report on the detailed characterization of RecA protein in the foodborne pathogen L. monocytogenes. Materials and methods 5

Biochemicals, bacterial strains, enzymes and DNA The chemicals used in this study were of analytical or molecular biology grade purchased from Merck, Kgga, Germany. The restriction enzymes, pfu polymerase, and ligase were purchased from New England Biolabs. The genomic DNA purification kit was procured from Sigma-Aldrich, USA. The plasmid DNA purification kit, gel extraction kit, RNA isolation kit, and Ni2+-NTA resin were obtained from Qiagen. The cDNA synthesis kit was procured from Thermo Scientific. Fast-

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performance liquid chromatography columns for protein purification were procured from GE Healthcare. The thin layer chromatography (TLC)-phosphocellulose polyethylenimine sheets (PEI) were procured from Merck. The site-directed

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mutagenesis kit was obtained from Agilent. The wild-type L. monocytogenes was from ATCC, with accession number ATCC 13932. The M13 phage derived

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negatively supercoiled and circular single stranded DNA were prepared as

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previously described [28].

The cloning primers, including oligonucleotides (ODNs) for DNA substrate

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preparation were synthesized from Sigma Genosys, Singapore. ODN sequences are given in the Supplementary Table 1. The ODN 3, ODN 4, and ODN 5 were

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labeled at the 5’ end by using 6-FAM fluorescence-labeling. The complementary duplex substrates (80-mer) for the DNA strand exchange assay, were prepared by

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annealing ODN 1 and 2. To check the DNA binding activity of the L. monocytogenes LexA protein, a 32 bp Cheo box dsDNA was prepared by annealing ODN 6 with the 6-FAM-labeled ODN 5. The annealed mixture were subjected to electrophoresis on a 10% polyacrylamide gel (PAGE) in 44.5 mM Tris-borate buffer (pH 8.3) containing 1 mM EDTA at 150 V for 7 h to 80-mer duplex and 4 h for Cheo box duplex substrates.

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The annealed substrates were excised from the gel and eluted into a TE buffer containing (10 mM Tris-HCl (pH 7.5) and 1 mM EDTA). UV light and MMS treatment The L. monocytogenes were grown at 37 °C in a brain heart infusion (BHI) broth with continous shaking at 180 rpm. At the mid exponential phase (A600 = 0.6), methyl methane sulfonate (MMS) was added to the culture to a final concentration of

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0.04%. In a parallel experiment, the cells were exposed to ultraviolet (UV) fluorescent lamp at 180 J/m2 for 6 min. The cells were further incubated at 37 °C in the dark with gentle shaking. At different intervals, the samples was harvested and

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suspended in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol and, 2 mM phenylmethane sulfonyl fluoride (PMSF) and processed for

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Quantitative real-time PCR

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quantitative real-time PCR (qRT-PCR) and western blotting.

The qRT-PCR reactions were performed on untreated L. monocytogenes cells

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and on those exposed to UV irradiation or MMS. The L. monocytogenes cells were grown as described above and the harvested cells were suspended in a buffer containing 15 mM Tris-HCl (pH 8) and, 0.45 M sucrose, 8 mM EDTA with 10 mg/ ml

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lysozyme and incubated for 1 h at 4 °C. The cell suspension was subjected to

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centrifugation for 15 min at 13,000 rpm. The RNA was isolated and 1 µg of RNA was used for the synthesis of cDNA. 100 ng cDNA was used for the real-time PCR experiment (Quantum plus real-time PCR machine, Applied Biosystems); and the sequences of the primers ware as follows: for 16s RNA: 5′ primer, 5′CACGTGCTACAATGGATAG-3’; 3′ primer, 5′- AGAATAGTTTTATGGGATTAG-3’ and for recA: 5′ primer, 5’- TAAAGTAGCGCCACCATTCC-3’; 3′ primer 5’-

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ATCCACTTCAGCAGCCATATC-3’. The program used for PCR consisted of 10 min of initial denaturation at 95 °C, followed by 40 cycles at 95 °C for 15 s, primer annealing at 52 °C for 30 s, extension at 72 °C for 30 s followed by the melting curve. Western blot analysis The western blot analysis was performed as described previously [29]. In brief: 20 µg of the cell free extract was loaded onto SDS-PAGE and electroblotted

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onto a polyvinylidene fluoride (PVDF) membrane using a wet blotter (Bio Rad) at 100 V for 100 min. The non specific sites were blocked with a NET buffer containing 1 M NaCl, 0.5 mM EDTA (pH 8), 1 M Tris-HCl (pH 7.8), 0.5% BSA and 1% titron X-100,

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followed by washing with TBST buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl and 0.1% Tween 20). The blot was incubated with the primary antibody followed by

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washing and incubation with HRP-linked anti rabbit secondary antibody. The blots

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were washed and the immunoreactive proteins were visualized using a chemiluminescence kit. The image was captured in the UVITEC chemiluminescence documentation system. The bands were quantified using Gene tools (ver. 4.3.8) from

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Syngene software and plotted using GraphPad Prism (ver.6.0).

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Bioinformatics analysis:

The L. monocytogenes, E. coli, Staphylococcus aureus, Bacillus cereus, and

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Shigella flexneri recA sequences were retrieved from the UniProt database. The sequences were analyzed for multiple sequence alignment using the Clustal Omega series of program and visualized using the Jalview software. Isolation of L. monocytogenes recA

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The gene coding for L. monocytogenes recA was amplified from genomic DNA, in PCR reactions using the pfu polymerase. Two PCR primers were designed using the reported L. monocytogenes recA gene sequence. The 5′ and 3’ primers contained the restriction sites EcoRI and XhoI respectively. The nucleotide sequences ware as follows: 5′ primer, 5′ATATGAATTCATGAATGATCGTCAAGCGG-3′; 3′ primer, 5′ATATCTCGAGTTTCATCATCTAGTAAACTTAATGTTTCTTC-3’. The PCR products

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(~1100 bp) were purified using the PCR purification kit from agarose gel and digested with the EcoRI and XhoI to remove the flanking sequence. The 1100 bp

fragment was ligated into the vector pET22a(+) between the restriction sites EcoRI

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and XhoI, downstream of the phage T7 promoter. The recombinant plasmid was

designated as pLMR1 and transformed into E. coli DH5α cells. The identity of the

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clone was confirmed by nucleotide sequencing as well as restriction mapping.

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Site-directed mutagenesis of L. monocytogenes recA A point mutation at 70th residue of lysine to arginine in L. monocytogenes recA was introduced by site directed mutagenesis using the kit obtained from Agilent

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(Catalog 200515). The protocol followed was as described by the manufacturer. To introduce point mutation, the primer was designed as follows 5’-

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CCAGAGAGTTCCGGTCGTACAACTGTTGCGCTTC-3’. The identity of the clone L.

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monocytogenes recA mutant gene was confirmed by restriction mapping and nucleotide sequencing and designated as pLMR1K70A. Isolation of L. monocytogenes lexA gene The L. monocytogenes lexA gene was amplified in a single PCR reaction from the genomic DNA of L. monocytogenes using the pfu polymerase. The primers were designed as per the reported gene sequence. The primer sequences are as follows:

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5’ primer, 5’-ATATGGATCCATGAAAATATCTAAACGCCAAC-3’; 3’ primer 5’ATATCTCGAGTGCGAATATCTCTATAAAGCCC-3’. The 5’ primer contained BamHI and the 3’ primer contained the XhoI restriction sites. Around 615 base-pair PCR product was purified through the PCR purification kit and both the PCR product and vector pET28a(+) were kept for double digestion with the respective restriction enzymes. The digested product was ligated into the vector pET28a(+) in between the two restriction sites BamHI and XhoI. The recombinant plasmid was confirmed by

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restriction mapping and nucleotide sequencing and designated as pLMLEXA. Purification of LmRecA protein:

The pLMR1 recombinant plasmid was subsequently transformed into the E.

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coli STL2669 ΔrecA strain for protein overexpression.The recombinant LmRecA

protein was purified through the conventional method. The E. coli STL2669, ΔrecA

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cells carrying the plasmid pLMR1 were cultured in a Luria-Bertani (LB) broth

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supplemented with ampicillin to the final concentration of 100 µg ml-1, and grown at 37 °C. At the mid exponential phase (A600 = 0.6), induction was done by the addition of Isopropyl 1-thio-β-D-galactopyranoside (IPTG) to the final concentration of 0.1 mM

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and cells ware further grown for 4 h. The cells were harvested by centrifugation at 5,000 rpm for 15 min at 4 °C and the cell pellet was suspended in 50 mM Tris-HCl

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(pH 8) containing 10% (W/V) sucrose, flash frozen and stored at -20 °C until use.

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The pellet was thawed on ice overnight and lysed by sonication (Vibra Cell Sonicator, Sonics and Materials Inc, Danbury, CT, USA) in pulse mode. The lysed cells were clarified by centrifugation at 20,000 rpm for 1 h at 4 °C and, the supernatant was precipitated by the addition of polymin P (pH 7.9) to the final concentration of 0.6 % over 30 min with continuous stirring. The pellet was collected by centrifugation at 10,000 rpm for 30 min and dissolved in buffer A (20 mM Tris-HCl

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pH7.5, 10% sucrose, 0.1 mM EDTA and, 5 mM 2-mercaptoethanol) containing 0.2 M NaCl and stirred for 30 min. The suspension was centrifuged at 10,000 rpm for 30 min; then, the supernatant was discarded and the pellet was extracted with buffer A containing 0.7 M NaCl. The supernatant was precipitated by the addition of ammonium sulphate (0.28g/ml) for 60 min. The pellet obtained from centrifugation at 10,000 rpm for 30 min was re-suspended in 30 ml of buffer B (20 mM sodium phosphate (pH 6.5), 0.1 mM EDTA, 0.2 M NaCl, 10 % glycerol and 5 mM 2-

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mercaptoethanol). This step was repeated twice and the pellet obtained was suspended in buffer B and dialyzed against the same buffer (3 changes) over a

period of 18 h. The dialyzed protein was subjected to a phospho cellulose column

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equilibrated with the same buffer. The unbound fractions having the RecA protein were pooled and dialyzed against the buffer C (20 mM Tris-acetate (pH7.5), 10%

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glycerol and 5 mM 2-mercaptoethanol) over a period of 18 h with 3 changes. The

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RecA protein was micro-crystallized by the addition of spermidine acetate (pH 7.5) to the final concentration of 9 mM for 30 min and subjected to centrifugation at 10,000 rpm for 30 min. The pellet was dissolved in buffer C containing 1 M NaCl and passed

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through a Sephacryl S-200 gel filtration column. The fractions containing the RecA protein were pooled and dialyzed against a storage buffer (20 mM Tris-acetate pH

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7.5, 0.1 mM EDTA, 20 % glycerol and 5 mM 2-mercaptoethanol) over a period of 18

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h with 3 changes. The concentration of the protein was determined by the dyebinding method using BSA as an internal standard [30] and small aliquots of the RecA protein were stored at -80 °C. The purity of the protein is assayed by 10% SDS-PAGE followed by Coomassie blue staining. Purification of LmLexA protein

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The pLMLEXA was transformed in the E. coli BL21 (DE3) expression host for overexpression of the LmLexA protein. The overnight grown culture was inoculated into fresh LB media containing kanamycin to the final concentration of 50 μg ml-1. The culture was incubated at 37 °C till A600=0.6, induced by the addition of IPTG to the final concentration of 0.5 mM and incubated further for 4 h. The cells were harvested by centrifugation and the pellet was washed with buffer A containing TrisHCl, pH 7.5, 100 mm NaCl and 1 mM EDTA. The cell pellet was resuspended in

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buffer B containing 20 mM Tris-HCl (pH 8), 0.5 M NaCl, and 10% glycerol, flash frozen and stored at -80 °C. The thawed cells were lysed by sonication in pulse

mode. The cell lysate was clarified by centrifugation at 20,000 rpm for 60 min at 4

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°C. The supernatant was loaded onto a 5 ml Ni2+ -NTA column and extensively

washed with buffer B containing 20 mM imidazole. The bound protein was eluted

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using a linear gradient of 20 to 500 mM imidazole in buffer B. The fractions

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containing highly pure LmLexA were pooled and precipitated by 0.3 g/ml ammonium sulfate. The pellet was collected by centrifugation at 14,000 rpm for 30 min at 4 °C and dialyzed against buffer B. The dialyzed protein was passed through a Sephacryl

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S-100 column, which had been equilibrated with the same buffer. The fractions containing LmLexA were pooled and precipitated with ammonium sulfate (0.3 g/ml).

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The pellet was collected by centrifugation at 14,000 rpm for 30 min at 4 °C and

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dialyzed against the storage buffer containing 20 mM Tris-HCl (pH 8), 0.5 M NaCl, 1 mM EDTA, 20 % glycerol and 1 mM 2-mercaptoethanol for 12 h. The protein concentration was checked by dye binding dye-binding method using BSA as an internal standard [30] and, stored at -80 °C. The quality of the protein was assessed by 12.5 % SDS-PAGE followed by Coomassie blue staining. Electrophoretic mobility shift assay

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The assay was performed as described previously [9, 31]. Twenty µl of the reaction mixture contained 20 mM Tris–HCl (pH 7.5),1.4 mM DTT, 0.1 mM ATPγS, 10 mM MgCl2, and 10 µM M13 cssDNA or 1 μM 6-FAM-labeled 80-mer oligonucleotide (ODN 3) and increasing concentrations of the LmRecA protein. The reaction mixture was incubated at 37 °C (30 min with M13 cssDNA and 10 min with oligonucleotide), and the reaction was stopped by the addition of 2.5 μl of 10X gel loading solution (50% glycerol, 0.42% (w/v) xylene cyanol, and 0.42% (w/v)

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bromophenol blue). The oligo based samples were subjected to electrophoresis on 8% native PAGE and electrophoresed in 44.5 mM Tris-borate buffer at 80 V for 2 h. The gels were directly visualized using the Typhoon FLA-9500 phosphorimager (GE

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Healthcare). The bands were quantified using Gene tools from Syngene software

and plotted using the GraphPad Prism software. The M13cssDNA based samples

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were subjected to electrophoresis on ethidium bromide (EtBr) free 0.8% agarose gel

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in 89 mM TBE buffer pH 8.3 at 2 V/cm for 4 h. The gels were stained in EtBr solution (0.2 µg/mL) and visualized using the Uvitec Cambridge gel documentation unit. Displacement-loop assay

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The assay was performed as stated previously [32]. The twenty μl of the reaction mixture containing 20 mM Tris–HCl (pH 7.5),1 mM DTT, 3 mM ATP, 0.1

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mg/ml BSA, 10 mM MgCl2, 5 mM phosphocreatine, 10 U/ml phosphocreatine kinase

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and 1 μM 6-FAM-labeled 80-mer oligonucleotide (ODN 4). The reaction was incubated at 37 °C for 5 min; 1 μM of LmRecA was added and the incubation was continued for an additional 5 min. Finally, the reaction mixture was initiated by the addition of negatively supercoiled M13 dsDNA (40 μM). At different time intervals, the reaction was stopped by the addition of SDS and proteinase K to a final concentration of 0.1% and 0.2 mg/ml, respectively. The samples were subjected to

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electrophoresis on 0.8% agarose gel in 89 mM Tris-borate buffer at 2 V/cm for 4 h. The gels was directly visualized using the phosphorimager. The bands were quantified in Gene tools from Syngene software and plotted using GraphPad Prism. Three strand exchange assay The assay was performed as described previously [9, 32]. Briefly: 20 μL of the reaction mixture containing 20 mM Tris-HCl (pH 7.5), 3 mM ATP, 10 mM MgCl2 ,1 mM DTT, 5 μM ssDNA (ODN 3) and 2.5 μM RecA was incubated in the presence of

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an ATP regeneration system (5 mM phosphocreatine and 10 units/ml creatine phosphokinase) at 37 °C for 5 min. The strand transfer reaction was initiated by the addition of 10 μM cold duplex (annealed using ODN 1 and 2) and incubation was

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continued for an additional 10 min. The reaction was stopped by the addition of 2.5

μL of 5X stop buffer containing 5% SDS and 100 mM EDTA, followed by the addition

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of 1.5 μL of 10X gel loading solution. The samples were loaded onto 10% native

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PAGE and electrophoresed in 44.5 mM Tris-borate buffer at 150 V for 7 h. The reaction products were visualized by the phosphorimager. The bands were quantified using Gene tools from Syngene software and plotted using GraphPad

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Prism.

ATP hydrolysis assay

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The standard ATPase assay was performed as stated previously [32, 33] in a

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10 μL reaction mixture containing 25 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, 5 μM M13 cssDNA and, 2.5 μM RecA. The reaction was initiated by the addition of 2 mM [γ-32P] ATP and incubated for 30 min at 37 °C. The reaction was stopped by the addition of 25 mM EDTA and 1.8 μL aliquots were transferred onto TLC-phosphocellulose PEI sheets that, were developed with a solution containing 0.5 M lithium chloride, 1 M formic acid and 1mM EDTA. The TLC sheets were air-

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dried and exposed to a phosphor intensiflying screen for 6 h. The screen was scanned in a phosphorimager. The bands were quantified using Gene tools from Syngene software and plotted using GraphPad Prism. RecA co-protease assay The assay was performed as previously described [9, 32]. The reaction mixtures contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 1 mM DTT, 3 mM ATP or 0.1 mM ATPγS, 5 μM ssDNA (ODN1), and 2 μM RecA and incubated for 5 min at

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30 °C. The co-protease activity was initiated by the addition of 4 μM LmLexA protein and incubation was continued at 30 °C. At different time points, the reaction mixture was terminated by boiling in SDS-loading dye. The samples were then subjected to

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12.5% SDS−PAGE and stained with silver nitrate. The bands were quantified using

Exonuclease protection assay

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Gene tools from Syngene software and plotted using GraphPad Prism.

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The exonuclease protection assay was performed as described previously [34]. The reaction mixture contained 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mM ATPγS, 5 μM ssDNA (ODN 3), and increasing concentration of RecA protein. The

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reaction was incubated for 5 min at 37 °C, then ExoI (having 3’ to 5’ polarity), was added to the reaction and incubated for an additional 10 min. The reaction was

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stopped by the addition of 2.5 μL of 5X stop buffer followed by the addition of

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proteinase K for deproteinization and incubated for 15 min. Finally a 10X gel loading solution was added and the reaction products were subjected to electrophoresis on a 10% native PAGE in 44.5 mM Tris-borate buffer at 80 V for 90 min. The gel was directly visualized in the phosphorimager.

Results

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Sequence comparison of RecA proteins The bacterial RecA protein is conserved and consists of three major domains: a central core domain flanker by, a small N terminal, and a large C terminal domain. The core domain is highly conserved among all bacterial species; it includes DNA binding sites and an ATP binding domain (walker A box) and is, also known as Ploop (G/A)XXXXGK(T/S) [35]. The N-terminal domain is short and mostly facilitates the RecA-ssDNA nucleoprotein filament formation by monomer-monomer

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interaction. The large C-terminal domain is poorly conserved throughout bacterial species, varies in length and, mostly consists of a high concentration of negatively charged residues. The RecA family of proteins is conserved in its active site

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architecture and overall structure among bacterial species. The L. monocytogenes

gene products that are required for DNA repair and genetic recombination and their

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orthologous proteins present in the prototype E.coli and different foodborne

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pathogens are given in Supplementary Table 2. The multiple sequence alignments engendered using Clustal Omega (Supplementary Figure 1) show that L. monocytogenes RecA shares 58 – 80 % identity and 75 – 92 % similarity with the E.

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coli, S. aureus, B. cereus, and S. flexneri RecA homologs. L. monocytogenes RecA expression in response to DNA damaging agents

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One of the mechanisms by which organisms overcome the stress of DNA

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damage by genotoxic agents is upregulation of the expression of DNA repair genes. Here, to determine the expression of LmRecA also increases in response to DNA damage, L. monocytogenes cultures were exposed to different DNA-damaging agents such as MMS and UV radiation. Following DNA damage, cells were harvested at different time periods and recA expression was chked by qRT-PCR as described in Materials and methods.

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Results show that L. monocytogenes recA mRNA levels increased 2.5-fold at 4 hours following DNA damage (Fig.1A). For UV-treated cells, mRNA levels declined to 1.5-fold above the level seen in untreated cells (Fig. 1B) while those in MMStreated cells remained high. To determine if protein levels also increased in response to DNA damage, the same cells were lysed and the cell free lysate was subjected to western blot analysis. A similar result was observed in the western blot analysis (Fig. 2C); the, quantified data (Fig.2D) showed that the RecA protein expression

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level increased in response to MMS and UV, compared to the untreated cells. The results clearly show that the recA gene product is inducible to DNA damaging agents.

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Purification of L. monocytogenes RecA, RecA K70A and LexA proteins To investigate the functional characteristics of LmRecA, we cloned,

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expressed, and purified the LmRecA and mutant LmRecA K70A proteins. The

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coomassie stained, 10 % SDS-PAGE gels show that the purified protein migrates as a single band corresponding to its expected molecular weight of 38 kDa (Fig. 2A, C).

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The identity of the proteins was confirmed by immunoblot analysis using anti-RecA antibody (Fig. 2B, D). Further, to ascertain the RecA coprotease function, we purified

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the L. monocytogenes LexA (LmLexA) protein through affinity chromatography as shown in 10% SDS-PAGE (Fig. 2E). The identity of the protein was confirmed

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through western blot analysis with anti-His antibody (Fig. 2F). The purified proteins were free from exo- and endonuclease contamination (data not shown). DNA binding properties of L. monocytogenes RecA, LmRecAK70A and LexA proteins To understand the DNA-binding properties of the recombinant LmRecA protein, we performed an elecrphoretic mobility shift assay (EMSA) in the presence 17

of a 6-FAM labeled 80-mer oligonucleotide as described under Materials and methods. The experimental design is shown in Fig. 3A. As shown in Fig. 3B and C, LmRecA binds to ssDNA in a concentration dependent manner and forms a stable DNA-protein complex as evidenced by the retardation of product movement in the gel than free DNA. However, the binding affinity of mutant LmRecAK70A is less compared to the wildtype protein. Further, we observed that with LmRecA in lower protein concentrations, there is a less well defined DNA protein complex and, the

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presence of a smear between the most-retarded band and free DNA in the gel. This indicates that either there is formation of multiple DNA protein complex species or

dissociation of the DNA protein complex during electrophoresis. To check the RecA

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binding to large plasmid substrates, we performed EMSA using M13 cssDNA. As shown in Fig S2A, LmRecA also binds to M13 cssDNA and there is a significant

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increase in DNA protein complex as the protein concentration is increased. The

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binding ability of LmRecA to dsDNA was also observed using a 6-FAM labeled linear duplex. S Fig. 2C shows, a weaker binding affinity of LmRecA towards linear duplex and the Fig. 2D show that LmRecA K70A is incapable to bind to duplex. Further, the

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EMSA was performed with the LmLexA protein using the Cheo-box dsDNA and 32 bp nonspecific random sequence dsDNA. As S Fig. 3A and B show, LmLexA binds

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specifically to the Cheo-box and does not show any binding affinity to the

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nonspecific/random DNA.

Displacement-loop formation by L. monocytogenes RecA During the early stages of homologous recombination, the RecA presynaptic

filament binds to dsDNA, locates the site of homology and facilitates localized exchange of strands resulting in the formation of a D-loop. To determine if LmRecA possesses this activity, a D-loop assay using a 6-FAM-labelled 80-mer

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oligonucleotide was paired with homologous negatively supercoiled DNA. The experimental design is shown in Fig. 4A. The protein was initially incubated with 6FAM labeled ssDNA and allowed to form active nucleoprotein filaments followed by the addition of negatively supercoiled dsDNA; the samples was taken out at different time periods as described in Materials and methods. As shown in Fig. 4B, LmRecA catalyzed the D-loop formation as the product migrated slowler than free DNA in the gel. The maximum product formation by LmRecA was within 10 min of incubation

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and reached plateau. The D-loop formed by LmRecA was stable and resistant to dissociation even after incubation of 30 min.

Strand transfer promoted by LmRecA and LmRecAK70A

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To gain insights into the mechanistic aspects of DNA strand transfer promoted by the LmRecA protein, we used an in vitro three-strand exchange assay using FAM

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labeled short oligonucleotide substrates. The assay involved paired substrates, i.e.,

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6-FAM labeled 80-mer ssDNA and 80-mer complementary cold duplex. The experimental design is shown in Fig. 5A. The time course experiment showed that LmRecA shows more than 80% heteroduplex formation after 10 min of incubation

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(Fig. 5B and 5D). Further, a strand exchange activity was performed with the LmRecAK70A protein with the same conditions as optimized for the LmRecA protein.

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As Fig. 5C and 5D show, only 50 % heteroduplex formation is seen after 20 min of

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incubation, suggesting that the reduced ATPase activity of this protein limits the strand exchange activity. DNA dependant ATPase activity of L. monocytogenes RecA The ATPase activity tightly regulates the RecA nucleoprotein filament formation to promote strand transfer activity in homologous recombination. Here we performed an ssDNA dependent ATP hydrolysis activity. The assay was

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accomplished as stated in Materials and methods; the RecA nucleoprotein filament hydrolyzed the [γ-32P] ATP in the reaction and released 32Pi which was separated using TLC. The experimental design is shown in Fig. 6A. As shown in Fig. 6B with increased concentrations of the RecA protein, the ATPase activity of LmRecA increased and reached a plateau after 2 µM with 40% product formation. The time course experiment showed that LmRecA reaches maximum activity after 30 min and reaches a plateau (Fig. 6E). On the other hand, LmRecA K70A did not show any

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significant product formation (Fig. 6C, F) with an increased concentration of protein or an increase in the incubation time period. Next the kinetic parameters of ATP

hydrolysis for the LmRecA protein were determined by nonlinear regression fitting to

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the Michaelis-Menten equation with increased concentrations of ATP in the presence M13 cssDNA. The Fig. 6H show, LmRecA with an apparent KM of 111.6 µM,

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whereas the extrapolated Vmax was 5.595 µmol min-1L-1, which corresponds to a kcat

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value of 11.189 min -1. Co-protease activity of LmRecA

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During severe DNA damage, the SOS cellular response is activated through transcriptional derepression, resulting from LexA cleavage. The LexA protein

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undergoes autoproteolysis with the help of the RecA nucleoprotein filament as ‘coprotease’. This event can be scored in vitro by monitoring LexA cleavage assay.

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The scheme of the experiment design is shown in Fig. 7A. To gain insight into the LmRecA biochemical functions we performed LexA cleavage assay as described in Materials and methods. The presence of RecA nucleoprotein filaments leads to autocatalysis of the LexA repressor, resulting in two peptides/products designated as P1 and P2. As shown in Fig. 7B and C, LmRecA readily catalyzes the reaction in the presence of both ATPγS and ATP; the reaction reaches its maximum activity within 20

15 min and formes a plateau till 60 min. The quantified data (Fig. 7D) shows a slight increase in P1 and P2 formation in the presence of ATPγS, indicating stable nucleoprotein filament formation by the RecA protein compared to ATP. Exonuclease Protection Assay We observe presynaptic filament protection activity of the RecA protein in vitro. As described in Materials and methods. The scheme of the experimental design is shown in Fig. 8A. The 5’- 6 FAM-labeled oligonucleotide was incubated

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with ExoI in the presence or absence of the RecA protein.. As shown in Fig. 8B, in the absence of RecA, there is a complete degradation of DNA by the ExoI activity.

However, with increased concentrations of LmRecA in the reaction, the degradation

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filament from degradation by ExoI activity.

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of DNA reduced. This suggests that the RecA protein protects the presynaptic

Discussion

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During recombination, RecA binds to ssDNA and forms RecA ssDNA filament, an initial crucial step needed for genetic repair, recombination as well as SOS

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induction. In the present study, we observed that the L. monocytogenes recA expression increased between within 2 h of exposure to the DNA damaging agents,

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MMS and UV, whereas, in other organisms such as Mycobacterium tuberculosis and Mycobacterium smegmatis the optimum RecA induction is seen after 24 h and 5 h of

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exposure respectively in response to mitomycin C [36]. Upon exposure to mytomycin C, the Bacillus subtilis RecA was fully expressed within 60 and 90 min [37-38]. This indicates that the L. monocytogenes RecA induction kinetic response to DNA damaging agents bears some resemblance to other gram positive bacteria, such as M. smegmatis and B. subtilis. Further, the doubling time of L. monocytogenes is around 45 min, which suggests that (consistent with previous observations), the 21

kinetics of RecA expression in response to DNA-damaging agents depends on the rate of replication of chromosomes of the organism [36]. In general, the RecA proteins are loaded onto replicon or damage induced ssDNA and form active nucleoprotein filaments. In this study, we examined the ability of LmRecA binding to different substrates such asa a large plasmid substrate M13 cssDNA, small 80-mer ssDNA and duplex. We observed that LmRecA robustly binds to both short and large ssDNA substrates indicating the formation of active

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nucleoprotein filament by LmRecA; it has less binding affinity towards dsDNA,. Further, the LmRecA active nucleoprotein filaments also catalyzes D-loop formation. The D-loop formed by LmRecA is quite stable and resistant to dissociation even after

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incubation of 30 min.

The DNA strand exchange reaction is the central feature of the HR process,

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catalyzed by RecA proteins and its homologs. In the initial stage of the strand

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exchange reaction, RecA proteins are loaded to the damage induced single stranded DNA and form long filament of DNA/RecA complex. Further, the RecA-ssDNA filament binds to the complementary double stranded DNA and finds a homologous

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sequence to drive strand exchange [39, 40]. In this study, LmRecA catalysed the strand transfer activity kinetically slower than the earlier reported RecA proteins.

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After 10 min of incubation it showed complete product formation whereas earlier

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reported prototype E. coli RecA, M. smegmatis RecA and M. tuberculosis RecA catalyzed the reaction within 30 s of incubation [9]. Further, in our previous studies, we observed that the LmRecA plasmid based strand exchange activity depends on the presence of the L. monocytogenes single stranded DNA binding protein1 (SSB1) [41]. Recent studies have suggested that during the recombination process, the ATP hydrolysis activity by the RecA nucleoprotein filament converts the high affinity DNA

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binding from RecA-ATP to the lower affinity form RecA-ADP [42]. This occurrence prevents the accumulation of the unwanted complexes which are produced by the direct binding of RecA to the undamaged dsDNA region. In our study, we also observed that in the presence of ssDNA, LmRecA effectively hydrolyzes ATP. Previously, it has been demonstrated that the substitution of lysine residue with arginine in E. coli RecA inhibits all kinds of RecA activities in vivo [43]. Comparatively, the purified mutant protein has reduced ATP hydrolysis and lack of

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complete plasmid-based strand exchange activity; it can catalyze the strand exchange reaction activity upto 1.5 kilobase only in the presence of dATP and not

ATP [44]. In this study, replacing lysine residue with arginine in the LmRecA protein

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also reduced the ATPase activity but it catalyzed the strand exchange reaction with

50% product formation in comparison with the wild-type protein with 80-mer ssDNA

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oligonucleotides. Consistent with earlier observations, this study also suggests that

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ATP hydrolysis is not essential for the strand transfer activity of the RecA protein [4446]. The ATPase activity of the RecA nucleoprotein filament including both ATP binding and hydrolysis is essential to remove the excessive RecA monomer

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assembly to the ssDNA gap [39]. Further, it has also been shown that the non filament forming RecA dimer can effectively recognize sequence homology and

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catalyze homologous formation [47]. The same study also suggested that the ATP

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hydrolysis activity of the RecA nucleoprotein filament may also be involved in the selection of recombination partners at the time of homologous recombination [47]. The DNA damage in organisms triggers the activation of the SOS response

pathway through the RecA coprotease activity. Approximately 40 genes are repressed by the LexA repressor during normal cell growth. Upon DNA damage, the RecA protein leads to the formation of active nucleoprotein filaments on ssDNA at

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the site of the damaged DNA and results in autocatalysis of the LexA repressor, which activates the SOS response [48-50]. An earlier study with L. monocytogenes also revealed that in response to heat shock, there is an increase in the expression of the heat-shocked proteins that leads to activation of the SOS response [51]. In the present study, we also observed that the LexA protein readily undergoes autocatalysis in the presence of active LmRecA nucleoprotein filaments, indicating that the regulation of SOS response by the RecA protein is conserved in L.

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monocytogenes. Further, it has also been shown that the ∆recA L. monocytogenes cells are sensitive to heat, acid exposure and H2O2 due to inactivation of the SOS response in the absence of the RecA protein [52,53].

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A recent study showed that during DSB, in vivo DNA degradation is regulated by

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the RecA protein by association with ssDNA through inhibition of the exonuclease activity. The RecA protein preserved the ssDNA 39 tail that is crucial for controlling

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the extent of the DSB processing [54]. In the present study, we also observed that the RecA protein protects the filament from in vitro degradation by inhibiting the

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exonuclease activity. Altogether, this is the first detailed study on the functional characterization of the RecA protein from L. monocytogenes and provides

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compelling evidence that the recA gene product is conserved and performs canonical functions. In summary, the findings reported in this paper on the RecA

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protein advances the understanding of the conservation of RecA functions across bacterial species. Conclusions In our study, we observed observed that L. monocytogenes RecA is expressed more than two-fold in response to stress conditions such as DNA-

24

damaging agents (MMS and UV). The purified LmRecA protein robustly binds to ssDNA in a sequence non-specific manner. The RecA forms D-loops, catalyzes the strand exchange hallmark activity and also hydrolyzes ATP. However, the mutant LmRecAK70A protein showed reduced strand exchange activity and failed to hydrolyze ATP as lysine residue of the walker A motif takes part in the ATP hydrolysis activity of RecA protein. The wild-type LmRecA protein cleaves the LexA protein and protects the RecA presynaptic filament from exonuclease I activity.

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Taken together, these results provide a detailed report on the functional characterization of the foodborne pathogen L. monocytogenes RecA protein and elucidate important aspects of RecA promoted activities.

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Author contributions: D.O. and K.N.P. conceived the study. D.O. performed the

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experimental work. K.N.P. and D.O. analyzed the data and wrote the paper. Both authors confirm that they have read and approved the final manuscript.

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the contents of this article.

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Conflict of interest: The authors declare that they have no conflicts of interest with

Acknowledgments

This work was supported by the Science and Engineering Research Board

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(SERB), Department of Science & Technology, Government of India through its

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Early Career Research Award (ECR/2016/000445) to K.N.P. Debika Ojha is a recipient of DST-INSPIRE (Innovation in Science Pursuit for Inspired Research) fellowship in 2014. The authors are grateful to the Director, CSIR-CFTRI for support and providing the in-house start-up grant; MLP-0181 and for partial support from the CSIR-12th five year plan project ‘Advance Facility for Research in Molecular Nutrition’ (BSC-0404) to K.N.P. The authors thank Professor Piero R. Bianco, University at

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Buffalo, Buffalo, USA, for critically reading the draft of this manuscript. The E. coli STL2669 ∆recA strain was a gift from Professor Michael M. Cox, University of Wisconsin-Madison, Wisconsin, USA. The authors thank Professor. K. Muniyappa, Department of Biochemistry, Indian Institute of Science, Bangaluru for allowing them to conduct radioactivity experiments and providing the polyclonal anti-

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Mycobacterium tuberculosis RecA antibody.

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References 1. Michel B, Flores MJ, Viguera E, Grompone G, Seigneur M, Bidnenko V. Rescue of arrested replication forks by homologous recombination. Proc Natl Acad Sci 2001; 98:8181–8188. https://doi.org/10.1073/pnas.111008798. 2. Lusetti SL, Cox MM. The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu Rev Biochem 2002; 71:71–100. https://doi.org/10.1146/annurev.biochem.71.083101.133940.

ro of

3. Krejci L, Altmannova V, Spirek M, Zhao X. Homologous recombination and its regulation. Nucleic Acids Res 2012; 40: 5795–5818. https://doi.org/10.1093/nar/gks270.

-p

4. Bianco PR, Tracy RB, Kowalczykowski SC. DNA strand exchange proteins: A

re

biochemical and physical comparison. Front Biosci 1998; 3: 570–603. 5. Cox MM. Regulation of bacterial RecA protein function. Crit Rev Biochem Mol

lP

Biol 2007; 42:41–63. https://doi.org/10.1080/10409230701260258. 6. Kowalczykowski SC. An Overview of the Molecular Mechanisms of

na

Recombinational DNA Repair. Cold Spring Harb Perspect Biol 2015; 7:a016410. https://doi.org/10.1101/cshperspect.a016410.

ur

7. Le S, Chen H, Zhang X, Chein J, Patil KN, Muniyappa K, Yan J. Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in

Jo

Mycobacterium tuberculosis. Nucleic Acids Res 2014; 42:11992–11999. https://doi.org/ 10.1093/nar/gku899.

8. Haruta,N, Yu X, Yang S, Egelman E H, Cox MM. A DNA pairing-enhanced conformation of bacterial RecA proteins. J Biol Chem 2003; 278:52710-52723. https://doi.org/10.1074/jbc.M308563200.

27

9. Patil KN, Singh P, Muniyappa K. DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments. Biochemistry 2015; 50: 300– 311. https://doi.org/10.1021/bi1018013. 10. Ngo KV, Molzberger ET, Chitteni-Pattu S, Cox MM. Regulation of Deinococcus radiodurans RecA protein function via modulation of active and inactive nucleoprotein filament states. J Biol Chem 2013; 288: 21351-2136.

ro of

https://doi.org/10.1074/jbc.M113.459230. 11. Windgassen TA, Wessel SR, Bhattacharyya B, Keck JL. Mechanisms of bacterial DNA replication restart. Nucleic Acids Res 2018; 46: 504–519.

-p

https://doi.org/10.1093/nar/gkx1203.

12. Anderson DG, Churchill JJ, Kowalczykowski SC. A Single Mutation,

re

RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by

lP

RecBCD Enzyme. J Biol Chem 1999; 274:27139–27144. https://doi.org/10.1074/jbc.274.38.27139

13. Bell JC, Kowalczykowski SC. RecA: Regulation and Mechanism of a Molecular

na

Search Engine. Trends Biochem Sci 2016; 41: 491–507. https://doi.org/10.1016/j.tibs.2016.04.002.

ur

14. Leite WC, Galvao CW, Saab SC, Iulek J, Etto RM, Steffens MB, Chitteni-Pattu

Jo

S, Stanage T, Keck JL, Cox MM. Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament. PLoS One 2016; 11: e0159871. https://doi.org/10.1371/journal.pone.0159871.

28

15. Howard-Flanders P, West SC, Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature 1983; 309: 215–219. https://doi.org/10.1038/309215a0. 16. De Vlaminck I, van Loenhout MT, Zweifel L. Mechanism of homology recognition in DNA recombination from dual-molecule experiments. Mol Cell 2012; 46: 616–624. https://doi.org/10.1016/j.molcel.2012.03.029. 17. Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC. Comparative

ro of

gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 2001; 158 : 41-64. PMCID:PMC1461638.

18. Fernandez De Henestrosa AR, Ogi T, Aoyagi S, Chafin D, Hayes JJ, Ohmori H,

-p

Woodgate R. Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol Microbiol 2001; 35: 1560-1572.

re

https://doi.org/10.1046/j.1365-2958.2000.01826.x

lP

19. Little JW, Mount DW. The SOS regulatory system of Escherichia coli. Cell 1982; 29: 11-22. https://doi.org/10.1016/0092-8674(82)90085-X. 20. Petrova V, Satyshur KA, George NP, McCaslin D, Cox MM, Keck JL. X-ray

na

crystal structure of the bacterial conjugation factor PsiB, a negative regulator of RecA. J Biol Chem 2010; 285: 30615-30621.

ur

https://doi.org/10.1074/jbc.M110.152298.

Jo

21. Gray MJ, Freitag NE, Boor KJ. How the bacterial pathogen Listeria monocytogenes mediates the switch from environmental Dr. Jekyll to pathogenic Mr. Hyde. Infect Immun 2006; 74: 2505-2512. https://doi.org/10.1128/IAI.74.5.2505-2512.2006.

29

22. Swaminathan B, Gerner-Smidt P. The epidemiology of human listeriosis. Microbes and Infect 2007; 9: 1236-1243. https://doi.org/10.1016/j.micinf.2007.05.011. 23. Veen SVD, Moezelaar R, Abee T, Wells-Bennik MH. The growth limits of a large number of Listeria monocytogenes strains at combinations of stresses show serotype- and niche-specific traits. J Appl Microbiol 2008; 105: 1246–1258. https://doi.org/10.1111/j.1365-2672.2008.03873.x.

ro of

24. Poyart-Salmeron C, Carlier C, Trieu-Cuot P, Courvalin P, Courtieu AL. Transfer-able plasmid-mediated antibiotic resistance in Listeria monocytogenes. Lancet 1990; 335: 1422–1426. https://doi.org/10.1016/0140-6736(90)91447-I.

-p

25. Noll M, Kleta S, Al Dahouk S. Antibiotic susceptibility of 259 Listeria

monocytogenes strains isolatedfrom food, food-processing plants and human

re

samples in Germany. J Infect Public Health 2018, 11:572–577.

lP

https://doi.org/10.1016/j.jiph.2017.12.007.

26. Lee AM, Wigle TJ, Singleton SF. A complementary pair of rapid molecular screening assays for RecA activities. Anal Biochem 2007; 367: 247–258.

na

https://doi.org/10.1016/j.ab.2007.04.021.

27. Nautiyal A, Patil KN, Muniyappa K. Suramin is a potent and selective inhibitor of

ur

Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a

Jo

potential target for antibacterial drug discovery. J Antimicrob Chemother 2014; 69:1834–1843. https://doi.org/10.1093/jac/dku080.

28. Cunningham RP, DasGupta C, Shibata T, Radding CM. Homologous Pairing in Genetic Recombination: recA Protein Makes Joint Molecules of Gapped Circular DNA and Closed Circular DNA. Cell 1980; 20: 223-235. https://doi.org/10.1016/0092-8674(80)90250-0.

30

29. Davis EO, Jenner PJ, Brooks PC, Colston MJ, Sedgwick SG. Protein splicing in the maturation of M. Tuberculosis RecA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 1992; 71: 201-210. https://doi.org/10.1016/0092-8674(92)90349-H. 30. Bradford MM. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem J 1976; 72 ; 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.

ro of

31. Ganesh N, Muniyappa K. Mycobacterium smegmatis RecA protein is structurally similar but functionally distinct from Mycobacterium tuberculosis RecA. Proteins: Struc Func & Genet 2003; 53: 6-17.

-p

https://doi.org/10.1002/prot.10433.

32. Patil KN, Singh P, Harsha S, Muniyappa, K. Mycobacterium leprae RecA is

re

structurally analogous but functionally distinct from Mycobacterium tuberculosis

lP

RecA protein. Biochimica. et. Biophysica. Acta. 2011; 1814: 1802–1811. https://doi.org/10.1016/j.bbapap.2011.09.011. 33. Ganesh N, Muniyappa K. Characterization of DNA strand exchange promoted

na

by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA. Biochemistry 2003; 42: 7216-7225.

ur

https://doi.org/10.1021/bi0340548.

Jo

34. Chang HY, Liao CY, Su GC. Sheng-Wei Lin§, Hong-Wei Wang, and Peter Chi. Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1. J Biol Chem 2015; 290:9863–19873. https://doi.org/10.1074/jbc.M115.666289. 35. Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the α-and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring

31

enzymes and a common nucleotide binding fold. EMBO J 1982; 1: 945-95. PMCID: PMC553140. 36. Papavinasasundaram KG, Anderson C, Brooks PC, Thomas N A, Movahedzadeh F, Jenner PJ, Colston MJ, Davis EO. Slow induction of RecA by DNA damage in Mycobacterium tuberculosis. Microbiology 2001; 147: 3271– 3279. https://doi.org/10.1099/00221287-147-12-3271. 37. Lovett CM, Jr Love PE, Yasbin RE, Roberts JW. SOS-like induction in Bacillus

ro of

subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains. J Bacteriol 1988; 170: 1467–1474. https://doi.org/10.1128/jb.170.4.1467-1474.1988.

-p

38. Lovett CM, Jr Cho KC, O Gara TM. Purification of an SOS repressor from Bacillus subtilis. J Bacteriol 1993;175: 6842–6849.

re

https://doi.org/10.1128/jb.175.21.6842-6849.1993.

lP

39. Zhao B, Zhang D, Li C, Yuan Z, Yu F, Zhong S, Jiang G, Yang YG, Le XC, Weinfeld M. ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination. Cell Discov 2017: 3: 16053.

na

https://doi.org/10.1038/celldisc.2016.53.

40. Lusetti SL, Wood EA, Fleming CD, Modica MJ, Korth J, Abbott L, Dwyer DW,

ur

Roca AI, Inman RB, Cox MM. C-terminal deletions of the Escherichia coli RecA

Jo

protein characterization of in vivo and in vitro effects. J Biol Chem 2003; 278: 16372–16380. https://doi.org/10.1074/jbc.M212917200.

41. Ojha D, Greeshma MV, Patil KN. Expression, purification and biochemical characterization of Listeria monocytogenes single stranded DNA binding protein 1. Protein Expr Purif 2019; 161: 63-69. https://doi.org/10.1016/j.pep.2019.04.007.

32

42. Gataulin DV, Carey JN, Li J, Shah P, Grubb JT, Bishop DK. The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double stranded DNA. Nucleic Acids Res 2018; 46: 9510-9523. https://doi.org/10.1093/nar/gky748. 43. Konola JT, Logan KM, Knight KL. Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site. J Mol Biol 1994; 237: 20–34. https://doi.org/10.1006/jmbi.1994.1206.

ro of

44. Rehrauer WM, Kowalczykowski SC. Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP

hydrolysis but not joint molecule formation. J Biol Chem 1993; 268: 1292–1297.

-p

45. Chiaradia L, Lefebvre C, Parra J, Marcoux J, Burlet-Schiltz O, Etienne G,

Tropis M, Daffé M. Dissecting the mycobacterial cell envelope and defining the

re

composition of the native mycomembrane. Sci Rep 2017; 7: 12807.

lP

https://doi.org/10.1038/s41598-017-12718-4.

46. Cox JM, Tsodikov OV, Cox MM. Organized unidirectional waves of ATP hydrolysis within a RecA filament. PLoS Biol 2005; 3: e52.

na

https://doi.org/10.1371/journal.pbio.0030052. 47. Cunningham RP, DasGupta C, Shibata T, Radding CM. Homologous Pairing in

ur

Genetic Recombination: recA Protein Makes Joint Molecules of Gapped Circular

Jo

DNA and Closed Circular DNA, Cell 1980; 20: 223-235. https://doi.org/10.1016/0092-8674(80)90250-0.

48. Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 1994; 58: 401-465. PMCID: PMC372975.

33

49. Michel B. After 30 years of study, the bacterial SOS response still surprises us. PLoS Biol 2005; 3: e255. https://doi.org/10.1371/journal.pbio.0030255. 50. Cox MM. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol 2007; 8: 127–138. https://doi.org/10.1038/nrm2099. 51. Veen SVD, Hain T, Wouters JA, Hossain H, Vos WMD, Abee T, Chakraborty T. Wells-Bennik MH. The heat-shock response of Listeria monocytogenes comprises genes involved in heat shock, cell division, cell wall synthesis, and

https://doi.org/10.1099/mic.0.2007/006361-0.

ro of

the SOS response. Microbiology 2007; 153: 3593–3607.

52. Veen SVD, Schalkwijk SV, Molenaar D, Vos WMD, Abee T. Wells-Bennik MHJ.

-p

The SOS response of Listeria monocytogenes is involved in stress resistance and mutagenesis. Microbiology 2010; 156: 374–384.

re

https://doi.org/10.1099/mic.0.035196-0.

lP

53. Ojha D, Patil KN. (2019) p-Coumaric acid inhibits the Listeria monocytogenes RecA protein functions and SOS response: An antimicrobial target. Biochem Biophys Res Commun 2019; 517: 655-661.

na

https://doi.org/10.1016/j.bbrc.2019.07.093. 54. Đermić E, Zahradka D, Vujaklija D, Ivanković S, Đermić D. 3'

ur

Terminated Overhangs Regulate DNA Double-Strand

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Break Processing in Escherichia coli. G3 (Bethesda) 2017; 7: 3091-3102. https://doi.org/10.1534/g3.117.043521

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Figure Legends Figure 1. Expression levels of L. monocytogenes recA in response to DNA damaging agents. (A) and (B) graphical representation of transcriptional changes of L. monocytogenes recA in response to MMS and UV respectively. Each data set represents the mean and standard deviation from three independently isolated RNA samples. The levels of expression were determined and normalized to 16S ribosomal RNA expression and induction ratios (fold increase) calculated relative to the untreated control. (C) Western blot analysis of RecA protein after exposing to MMS and UV at different time interval as mentioned on top of images. (D) Graphical

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representation of western blot analysis. The data point represent the mean ± SE of three independent experiments.

Figure 2. Expression and purification profile of LmRecA, LmRecA K70A, and

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LmLexA. 10% SDS-PAGE analysis showing induced expression of LmRecA (A) and LmRecA K70A (C) and various stages of purification. About 10 µg (Lane 2, 3, 4) and 5 µg (Lane 5, 6, 7) protein has been loaded in gel and visualized by Coomassie blue

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staining. Lane 1 and 8, molecular mass marker; 2, uninduced cell lysate; 3, induced cell lysate; 4, 0.7M NaCl extract of polymin P pellet; 5, dialysate from

lP

phosphocellulose column; 6, gelfiltration input; 7, purified LmRecA/ LmRecA K70A protein. (B) and (D) Westernblot analysis of LmRecA and LmRecAK70A using antiMycoacterium tuberculosis RecA antibody. (E) 12.5% SDS-PAGE showing induced

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expression of LmLexA and various stages of purification. About 10 µg (Lane 2, 3) and 5 µg (Lane 4, 5, 6) protein have been loaded in gel. Lane 1 and 7, molecular mass marker as indicated on left side of gel image; 2, uninduced cell lysate; 3,

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induced cell lysate; 4, elute from Ni+-NTA matrix’ 5, input of gelfiltration column; 6, purified LmLexA protein. (F) Western blot analysis of LmLexA using anti-His

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antibody.

Figure 3. ssDNA binding activity of LmRecA. (A) Schematic depiction of the experimental design. 3 µM 6-FAM labeled oligonucleotide ssDNA was incubated in the absence (lane 1) and presence of increasing concentration of LmRecA protein (B) and LmRecA K70A (C) the concentration of protein was mentioned in the panel (lane 2 to 9). (D) graphical representation of binding of LmRecA and LmRecA K70A to

35

the ssDNA. The data point represent the mean ± SE of three independent experiments. Figure 4. D-loop formation promoted by LmRecA. The reaction was performed as described in “Materials and methods”. (A) Schematic depiction of experimental design. (B) 6-FAM labeled ssDNA incubated in the absence or presence of LmRecA; lane 1, absence of LmRecA; lane 2-9, presence of LmRecA protein with an increase in incubation time as mentioned in the panel. The position of substrates and reaction products were indicated at the left-hand side of the gel image. (C) Graphical representation of the extent of the formation of D-loops by LmRecA as a function of

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time. The data point represent the mean ± SE of three independent experiments. Figure 5. Strand exchange activity promoted by LmRecA and LmRecAk70A proteins. (A) Schematic depiction of the experimental design. Reactions were performed as described in “Materials and methods”. Lane 1, show the control K70A

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reaction performed in the absence of RecA, Lanes 2-9 (LmRecA (B); and LmRecA

(C)), shows strand exchange reactions carried out for 0.25, 0.5, 1, 2, 5, 10, 15,

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and 20 min, respectively. The positions ssDNA and heteroduplex was indicated on the left-hand side of the gel. (D) Shows the graphical representation of the extent of

lP

strand exchange, as a function of reaction time. The data point represent the mean ± SE of three independent experiments.

k70A.

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Figure 6. DNA dependent ATP hydrolysis catalyzed by LmRecA and LmRecA (A) Schematic depiction of the experimental design. Reaction products were

separated by TLC, visualized by autoradiography and quantified as described in

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“Materials and Methods”. ATPase activity of LmRecA (B) and LmRecA k70A (C) as a function of increasing amounts of RecA. Lane 1, shows control reaction in the

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absence of RecA; lane 2, shows in the presence of RecA but in the absence of ssDNA; lane 3-9, shows in presence RecA protein with increased concentration as mentioned in the panel. (D) Quantification of data obtained from (B) and (C) as a function of the increase in RecA. Kinetics of ATP hydrolysis catalyzed by LmRecA (E) and LmRecA k70A (F). Lane 1, shows control reaction in absence of RecA; lane 2, shows in the presence of RecA but in the absence of ssDNA; lane 3-9, increase in the incubation time as mentioned in the panel. (G) Quantification of data obtained from panels (E) and (F) as a function of time. (H) The rate ATP hydrolysis catalyzed 36

by LmRecA in the presence of ssDNA, was plotted as a function of ATP concentration. The inset shows the summary of kinetic parameters of LmRecA protein. The data point represent the mean ± SE of three independent experiments. Figure 7. Co-protease activity of LmRecA protein. The assay was performed as described in “Materials and Methods”. (A) Shows the schematic depiction of experimental design. The experiment was performed in the presence of ATPγS (B) and ATP (C). Lane 1, shows the SDS-PAGE standard molecular mass markers; lane 2, shows LmRecA; lane 3, shows LmLexA; lane 4, control reaction in the absence of DNA; lane 5–12, shows complete reaction mixtures incubated for 0.5, 1, 3, 5, 10, 15,

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30 and 60 min, in presence of ssDNA. The positions cleavage products (P1 and P2) are indicated right-hand side of the gel image. (D) shows the graphic representation of the amount of uncleaved LmLexA as a function of reaction time. The data point represent the mean ± SE of three independent experiments.

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Figure 8. Determination of RecA presynaptic filament protection against ExoI activity. (A) schematic depiction of the ExoI protection assay. The reaction was

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performed as described in “Materials and Methods”. The FAM label is denoted by the asterisk. (B) Lane1, shows the ssDNA in the absence of both ExoI and RecA

lP

proteins, Lane 2, shows control reaction presence of ExoI and absence of RecA protein, Lane 3-7, in the presence of LmRecA. The concentration of proteins was

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mentioned in the panel.

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Figures

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Figure 1.

37

B

MMS

C

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A

D

C

Time (h) 2 4 6

12

C

UV Time (h) 2 4 6

12

-p

RecA

lP

RecA

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ur

na

GAPDH

Figure 2.

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GAPDH

38

A

B

kDa 116 66 LmRecA

25 18 14 1

2

3

4

5

6

7

8

C

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45 35

D

-p

kDa 116

re

66

25

na

18 1

E

2

3

4

5

6 7

8

F

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kDa

LmRecAK70A

lP

45 35

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116 66 45 35

LmLexA

25 18 14 1

2

3

4

5 39

6

7

Figure 3.

A Nucleoprotein filament

6-FAM labeled ssDNA + RecA

B

D

0.1 0.2 0.4 0.8 1.2 1.6 2 3

-RecA

ssDNA LmRecA K70A (

-RecA

DNAprotein complex

C

ssDNA LmRecA (

Terminate

1

2 3

1 2

0.1 0.2 0.4 0.8 1.2 1.6 2

3

4 5 6

7

8 9

3

4 5

6

7

8

9

Figure 4.

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A

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Nucleoprotein M13 negatively + superhelical dsDNA filament

6-FAM labeled + RecA ssDNA

B

C

Time (min) 1.5 3 5 10 15

2

3

4

5

6

7

8

9

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1

na

D-loop

Free DNA

30

lP

-RecA

LmRecA 0.5 1

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Free DNA

40

D-loop

Figure 5.

A Nucleoprotein + dsDNA filament

6-FAM labeled + RecA ssDNA

B

C

1

Time (min) 2 5 10

15

20

-RecA

-RecA

LmRecA 0.25 0.5

Displaced Hetero duplex + ssDNA

9

1

0.25 0.5

LmRecA K70A Time (min) 1 2 5 10

15

20

Hetero duplex

1

2

3

4

5

6

7

8

2

3

4

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na

lP

re

-p

D

ro of

ssDNA

41

5

6

7

8

9

Figure 6.

A C -RecA -DNA

B -RecA -DNA

ADP + γ-32P Pi

Nucleoprotein + γ-32P ATP filament

ssDNA + RecA

LmRecA (µM)

.25 .5 .75 1 2 3 5

D LmRecA K70A (µM) .25 .5 .75 1 2 3

5

γ-32P Pi

Origin 1 2 34 5 6 7 8 9

F

LmRecA Time (min)

LmRecA K70A Time (min)

G

1 5 10 15 30 45 60

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1 5 10 15 30 45 60

-RecA -DNA

-RecA -DNA

E

1 2 3 4 5 6 7 8 9

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γ-32P ATP

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γ-32P Pi

γ-32P ATP

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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na

H

lP

Origin

42

Figure 7.

A Nucleoprotein + LexA filament

C

Time (min) 0.5 1

3 5 10 15 30 60

116 66 45 35 25 18 14

P1 + P2

LmRecA (ATP) Time (min) 0.5 1

3 5

10 15 30 60

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kDa

LmRecA (ATPγS)

Autoproteolysis

M LmRecA LmLexA -DNA

B

M LmRecA LmLexA -DNA

ssDNA + RecA

PI PII

2

3

4 5

6 7 8

1 2

9 10 11 12

3 4

5 6 7

-p

1

Jo

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na

lP

re

D

43

8

9 10 11 12

Figure 8.

A

*ssDNA + Exo I

1

2

3

-p

0.2 0.5 1

ro of

LmRecA (µM)

lP

*

re

5’

*

-RecA

B

5’

ssDNA

5’

*

5’

5’

2

3

4

5

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na

*

44

6

7