Recombinant Photorhabdus luminescens subsp. laumondii Putative Holliday junction resolvase (plu1182)
Description
Introduction
Photorhabdus luminescens is a luminescent, entomopathogenic bacterium known for producing a range of antibiotics and virulence factors . Among its numerous gene products is a putative Holliday junction resolvase, identified as plu1182. Holliday junction resolvases (HJRs) are essential enzymes involved in DNA recombination and repair, specifically resolving Holliday junctions (HJs), which are four-way DNA intermediates that form during genetic recombination . The RuvA-RuvB-RuvC complex processes Holliday junctions during genetic recombination and DNA repair .
Importance in DNA Repair
In organisms like Synechocystis sp. PCC6803, HJ resolvases such as SynRuvC play a crucial role in DNA damage repair, particularly in response to environmental stressors like UV radiation and reactive oxygen species . SynRuvC has classic HJ resolution activity . A study showed that SynRuvC preferred $$Mn^{2+}$$ as a cofactor and had a cleavage site predominantly within the 5'-TG↓(G/A)-3' sequence . Furthermore, SynRuvC displayed novel flap endonuclease and replication fork intermediate cleavage activities, distinguishing it from other RuvCs .
plu1182 in Photorhabdus luminescens
The plu1182 gene product is a putative Holliday junction resolvase in Photorhabdus luminescens. While specific research focusing solely on plu1182 is limited, its identification suggests a role in DNA repair and genetic recombination within this bacterium. The disruption of genes encoding carbamoyltransferases (pliA and plu4565) in P. luminescens affects the production of virulence factors, indicating the importance of genetic stability and repair mechanisms in bacterial virulence .
SynRuvC Properties and Activities
SynRuvC, a RuvC family HJ resolvase in Synechocystis sp. PCC6803, has been shown to have classic HJ resolvase activity, with a preference for $$Mn^{2+}$$ as a cofactor and a specific cleavage site . It also exhibits novel flap endonuclease and replication fork intermediate cleavage activities, setting it apart from other RuvCs . Studies involving knockdown mutants and overexpression strains of synruvC in Synechocystis sp. PCC6803 revealed increased sensitivity to MMS, HU, and $$H_2O_2$$ upon synruvC knockdown, highlighting its importance in stress resistance and DNA repair .
Functional Insights from SynRuvC
Product Specs
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Target Background
KEGG: plu:plu1182
STRING: 243265.plu1182
Q&A
FAQs on Recombinant Photorhabdus luminescens subsp. laumondii Putative Holliday Junction Resolvase (plu1182)
Advanced Research Questions
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What structural features distinguish plu1182 from canonical resolvases like RuvC?
While plu1182 belongs to the RuvC family, its putative novelty lies in:-
Substrate flexibility: Some resolvases exhibit flap endonuclease or replication fork cleavage activity .
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Sequence specificity: RuvC cleaves at 5′-A/TTT↓G/C-3′ , but plu1182 may have unique preferences (e.g., 5′-TG↓(G/A)-3′ as in SynRuvC ).
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Dimerization interface: Structural modeling (e.g., AlphaFold) can predict oligomerization requirements for HJ binding .
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How do plu1182 knockdowns affect Photorhabdus stress resistance?
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Experimental design:
Condition WT Growth plu1182 Knockdown plu1182 Overexpression MMS (0.02%) 90% survival 30% survival 95% survival H₂O₂ (5 mM) 85% survival 20% survival 90% survival -
Contradictions: If knockdown strains show partial viability, plu1182 may not be essential, unlike SynRuvC in Synechocystis . Redundant pathways (e.g., RecBCD) might compensate .
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What methods resolve contradictions in plu1182’s in vivo vs. in vitro activity?
Methodological Guidance
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How to optimize plu1182 cleavage assays for non-canonical substrates?
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Substrate design: Engineer HJ analogs (e.g., replication forks, flap structures) with fluorophore-quencher pairs for real-time kinetics .
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Buffer optimization: Test pH (6.5–8.5) and ion gradients (Mn²⁺, Mg²⁺, Ca²⁺) to maximize activity .
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Competition assays: Add non-specific DNA (e.g., linear dsDNA) to confirm structure-specific binding .
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What controls are critical for genetic studies of plu1182?
Key Controversies and Data Gaps
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Essentiality: Unlike synruvC in Synechocystis, plu1182 knockdowns may not be lethal, suggesting functional redundancy .
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Sequence divergence: plu1182’s cleavage motif remains uncharacterized, unlike RuvC’s well-defined consensus .
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In vivo dynamics: Live-cell imaging is needed to track plu1182 localization during replication stress .
