Recombinant Synechocystis sp. DNA replication and repair protein recF (recF)

What is the primary function of RecF protein in Synechocystis sp. PCC 6803?

RecF is a critical component of the RecF pathway involved in DNA recombinational repair, particularly for mending daughter strand gaps that occur during DNA replication. While most detailed characterization comes from E. coli models, the fundamental functions appear conserved in cyanobacteria like Synechocystis. The RecF pathway represents one of two major recombinational repair pathways in bacteria, alongside the RecBC pathway that repairs disintegrated replication forks .

In Synechocystis, RecF likely plays a crucial role in maintaining genomic integrity under various stress conditions, including those unique to photosynthetic organisms. The protein functions as part of a complex with RecO and RecR proteins to facilitate RecA loading onto single-stranded DNA at daughter strand gaps.

How does the RecF pathway operate in DNA repair mechanisms?

The RecF pathway follows a sequential process:

• Presynapsis: The RecF, RecO, and RecR proteins work together to prepare damaged DNA for homology search. The RecOR complex binds to SSB-complexed daughter strand gaps, potentially guided by the RecFR complex .

• Synapsis: RecO allows RecA polymerization on the SSB-complexed ssDNA. The RecA filament then locates an intact homologous duplex and pairs with it, enabling strand exchange .

• DNA synthesis: The pairing of damaged and intact DNA molecules allows gap filling by DNA polymerase, facilitated by topoisomerases - DNA gyrase relieves positive supercoils generated during strand invasion, while topoisomerase I relieves negative supercoils in the new duplex .

• Postsynapsis: Resolution of recombination intermediates occurs via the RuvABC resolvasome or RecG helicase, which removes Holliday junctions and the associated RecA filaments, completing the repair process .

How is recF expression regulated in cyanobacteria?

Unlike SOS-regulated repair genes, recF expression in cyanobacteria appears to be constitutive rather than strongly damage-inducible. This pattern aligns with observations of other recombinational repair proteins like RecG in E. coli, which shows no indication of enhanced expression during the SOS response .

In Synechocystis, recF expression may be influenced by circadian rhythms, given the significant role of circadian clock proteins in regulating metabolism and stress responses in this organism . The KaiABC-based oscillator and RpaA response regulator, which are central to circadian regulation in Synechocystis, might indirectly affect DNA repair systems including recF expression patterns during light/dark cycles.

What is the relationship between RecF-mediated DNA repair and photosynthetic metabolism in Synechocystis?

RecF-mediated DNA repair likely has specialized significance in photosynthetic organisms like Synechocystis due to several factors:

• Oxidative stress: Photosynthesis generates reactive oxygen species that can damage DNA, potentially increasing reliance on RecF-mediated repair mechanisms.

• Metabolic transitions: During transitions between photoautotrophic and heterotrophic growth, DNA replication patterns change, possibly affecting the types and frequency of DNA lesions that require RecF pathway repair .

• Circadian regulation: The deletion of circadian clock components (KaiAB1C1) or the response regulator RpaA in Synechocystis reduces growth in light/dark cycles and prevents heterotrophic growth in the dark . This suggests potential coordination between circadian rhythms, metabolism, and DNA repair systems including RecF.

Researchers investigating this relationship should consider experimental designs that compare recF activity under different light conditions, in various metabolic states, and in clock protein mutants.

How does DNA supercoiling affect RecF-mediated DNA repair in Synechocystis?

DNA topology significantly impacts DNA repair processes, including those mediated by RecF:

• DNA supercoiling levels in Synechocystis are regulated by a homeostatic control system involving topoisomerases . Manipulating this system through inducible overexpression or CRISPRi-based knockdown of topoisomerases affects genome-wide expression patterns .

• RecF-mediated repair involves extensive DNA manipulation requiring proper DNA topology. The daughter strand gap repair process specifically requires topoisomerase activity - DNA gyrase relieves positive supercoils while topoisomerase I relieves negative supercoils .

• Experimental approaches to study this relationship could include analyzing RecF repair efficiency in strains with altered supercoiling (through topA, gyrA, or gyrB manipulations) and examining how RecF activity correlates with supercoiling changes during different growth phases or environmental conditions.

How can CRISPRi technology enhance functional studies of recF in Synechocystis?

CRISPRi provides powerful advantages for studying recF function in Synechocystis:

• Tunable repression: Unlike knockout approaches, CRISPRi allows for controlled, partial repression of gene expression, which is particularly valuable for studying essential genes .

• Combinatorial studies: CRISPRi libraries in Synechocystis, as described in search result , enable simultaneous testing of multiple gene repressions, potentially revealing genetic interactions between recF and other repair or metabolic genes.

• Condition-specific requirements: CRISPRi can help determine the importance of recF under specific stress conditions by inducing repression at different experimental stages.

• Phenotypic enrichment: Similar to how researchers identified genes affecting lactate tolerance using CRISPRi libraries , this approach could identify conditions where recF repression provides advantages or disadvantages.

A methodological approach would involve designing sgRNAs targeting recF, creating strains with inducible dCas9 expression, and comparing phenotypes across various growth and stress conditions with different levels of recF repression.

What are effective methods for expressing and purifying recombinant Synechocystis RecF protein?

Optimization strategies:

• Codon optimization for the expression host

• Low-temperature induction (16-18°C) to enhance proper folding

• Co-expression with chaperones if misfolding occurs

• Testing multiple solubility tags and fusion partners

• Inclusion of DNA in purification buffers to stabilize DNA-binding proteins

Final purification should include size exclusion chromatography to ensure homogeneity, and activity assays to confirm proper folding and function.

How can researchers measure RecF-mediated DNA repair activity in vitro?

Biochemical assays:

• DNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) with ssDNA, gapped DNA, and duplex DNA substrates

  • Fluorescence anisotropy with labeled DNA substrates

  • Surface plasmon resonance to measure binding kinetics

• RecA loading assays:

  • ATP hydrolysis assays measuring RecA activation on SSB-coated ssDNA in the presence of RecF, RecO, and RecR

  • Strand exchange assays to measure RecFOR-facilitated RecA activity

• Reconstituted repair assays:

  • In vitro gap-filling assays using purified RecF, RecO, RecR, RecA, SSB, DNA polymerase, and topoisomerases

  • Direct visualization of repair intermediates using electron microscopy or single-molecule approaches

These assays should be performed under conditions that mimic the physiological environment of Synechocystis, accounting for its unique ionic requirements and potential regulatory factors.

What approaches can effectively characterize recF genetic interactions in Synechocystis?

• CRISPRi-based screening:

  • Create a library of strains combining recF repression with repression of other DNA repair genes

  • Test fitness under various DNA damaging conditions (UV, oxidative stress, replication inhibitors)

• Synthetic genetic array (SGA) analysis:

  • Systematically cross recF mutants with other Synechocystis mutants to identify synthetic lethality or suppression

  • Quantify growth phenotypes using automated imaging and analysis

• Transcriptome and metabolome analysis:

  • Compare RNA-seq profiles of wild-type and recF mutant strains under various conditions

  • Integrate with metabolomic data to identify metabolic signatures of recF deficiency

• Suppressor screening:

  • Identify secondary mutations that suppress recF mutant phenotypes

  • Sequence suppressor strains to identify the genetic basis of suppression

These approaches can reveal functional relationships between recF and other cellular systems, particularly those unique to cyanobacteria such as photosynthetic metabolism and circadian regulation.

How do researchers quantitatively assess RecF protein levels and localization in Synechocystis cells?

• Absolute quantification techniques:

  • Mass spectrometry-based approaches can provide absolute quantification of RecF protein levels per cell

  • Western blotting with purified RecF standards for calibration

  • Single-molecule counting using fluorescent protein fusions or antibody labeling

• Dynamics and localization:

  • Fluorescent protein tagging (ensuring functionality is preserved)

  • Time-lapse microscopy to track RecF localization during cell cycle and after DNA damage

  • Co-localization studies with replication machinery proteins

• Chromatin immunoprecipitation (ChIP):

  • Mapping RecF binding sites across the Synechocystis genome

  • ChIP-seq analysis before and after DNA damage to identify damage-dependent binding

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measuring RecF mobility within the cell under different conditions

  • Determining if RecF forms stable or transient complexes at repair sites

These quantitative approaches provide crucial insights into RecF function beyond what genetic studies alone can reveal, particularly regarding stoichiometry, dynamics, and spatial organization of repair processes.

How does RecF activity in Synechocystis differ between photoautotrophic and heterotrophic growth conditions?

Synechocystis can grow in both photoautotrophic and heterotrophic conditions, with significant metabolic differences between these states. Research indicates that:

• DNA repair requirements likely differ between light and dark conditions, as circadian clock proteins (KaiAB1C1) and the response regulator RpaA significantly affect the light/dark metabolic switch .

• Mutants lacking KaiAB1C1 or RpaA show reduced growth in light/dark cycles and cannot grow heterotrophically in the dark, suggesting metabolic imbalances that may impact DNA replication and repair requirements .

• The strongest metabolic changes in these mutants occur in the dark, including overaccumulation of 2-phosphoglycolate and altered carbon metabolism affecting pyruvate-derived amino acids .

Researchers should design experiments comparing RecF activity and importance across different growth modes, potentially using CRISPRi-based repression of recF under controlled conditions, followed by comprehensive phenotypic and metabolic analysis.

What is the impact of recF mutation on tolerance to environmental stressors in Synechocystis?

RecF-mediated DNA repair may be particularly important under specific stressors relevant to cyanobacteria:

• Oxidative stress: Pooled screening approaches similar to those used in search result could identify whether recF repression affects survival under oxidative stress conditions.

• Nutrient limitation: Synechocystis demonstrates complex metabolic responses to nutrient availability , potentially affecting DNA replication patterns and repair requirements.

• Chemical stressors: The approach used to identify that bcp2 repression increases tolerance to L-lactate by 49% could be adapted to test if recF repression affects tolerance to various chemical stressors.

• High light stress: Given that light intensity affects metabolic balance in Synechocystis, with factors like pmgA regulating photosystem content , recF may have specialized importance under high light conditions that increase DNA damage.

Experimental designs should incorporate metabolic analyses alongside survival measurements to connect RecF function to the broader cellular response to environmental challenges.

How conserved is RecF structure and function between Synechocystis and other bacterial species?

While the basic RecF pathway appears conserved across bacteria, important differences may exist:

• The core RecF pathway components (RecF, RecO, RecR) are present across diverse bacteria, but sequence conservation varies, potentially reflecting adaptations to different genomic architectures and environmental challenges.

• In E. coli, recF null mutations reduce viability to approximately the same degree as recBC mutations (about 85%) , but the relative importance of these pathways may differ in Synechocystis due to its unique metabolism and genome structure.

• The relationship between RecF and RecG may differ between species - in E. coli, recG mutations cause only moderate reduction in cell survival after UV or X-ray treatment , but this relationship might be altered in photosynthetic organisms experiencing different types of DNA damage.

Comparative genomic and structural analyses, complemented by cross-species functional complementation experiments, would help identify cyanobacteria-specific adaptations in RecF structure and function.

How does photorespiration in Synechocystis influence RecF-mediated DNA repair?

Photorespiration generates unique metabolic challenges in photosynthetic organisms that may interact with DNA repair systems:

• Synechocystis uses multiple pathways to metabolize 2-phosphoglycolate produced during photorespiration, including a phosphoserine pathway that functions as an auxiliary supply of serine .

• RubisCO oxygenase activity (approximately 3% of its carboxylase activity) creates metabolic flux through photorespiratory pathways , potentially generating specific types of DNA damage or metabolic stress.

• Mutations in circadian clock components lead to overaccumulation of 2-phosphoglycolate and RbcL protein, suggesting enhanced RubisCO activity in the dark - this metabolic imbalance may create unique requirements for DNA repair systems including RecF.

Researchers should investigate how alterations in photorespiratory flux (through environmental conditions or genetic manipulation) affect the importance and activity of RecF-mediated repair in Synechocystis.