Recombinant Treponema denticola Crossover junction endodeoxyribonuclease RuvC (ruvC)

Introduction to Recombinant Treponema denticola Crossover Junction Endodeoxyribonuclease RuvC (RuvC)

RuvC is an endodeoxyribonuclease that plays a crucial role in DNA repair and genetic recombination processes, particularly in resolving Holliday junctions . Holliday junctions are branched DNA structures that form during homologous recombination, a process essential for repairing double-strand breaks and ensuring genetic diversity . The RuvC protein is part of the RuvABC complex, which includes RuvA and RuvB proteins, all working together to process Holliday junctions .

In Treponema denticola, a periodontal pathogen, RuvC likely functions similarly in DNA repair and recombination. T. denticola is an oral spirochete associated with periodontitis, and understanding its DNA repair mechanisms can provide insights into its survival and pathogenicity . The "recombinant" designation indicates that the RuvC protein is produced using recombinant DNA technology, allowing for its isolation and study in controlled experimental settings.

Structure and Function of RuvC

The RuvC protein is an endonuclease, meaning it cleaves phosphodiester bonds within a DNA strand . Specifically, RuvC resolves Holliday junctions by cleaving two DNA strands at the junction point, leading to the separation of the recombining DNA molecules . This resolution is essential for completing homologous recombination and DNA repair.

Key aspects of RuvC's structure and function include:

• Nuclease Domains: Cas9 protein comprises an RuvC nuclease domain that cleaves a single DNA strand at a target sequence . The concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick .

• Subdomains: The RuvC domain comprises subdomains I, II and III, where domain I is located near the N- terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain .

Role in CRISPR-Cas Systems

RuvC is a component of the Cas9 enzyme, which cleaves DNA utilizing Rec-I/II, HNH, RuvC, and a bridge helix . The RuvC slices the other DNA strand and breaks the DNA into two strands (double-stranded DNA) . The Cas9 enzyme, acting as a molecular scissor protein, forms a complex with the sgRNA and directs the precise cutting at the desired location in the genome .

RuvC in DNA Repair Mechanisms

RuvC is involved in the cellular response and repair pathways for trapped topoisomerase complexes . Mutations affecting RuvABC activity result in increased sensitivity to certain antibiotics, highlighting the importance of Holliday junction resolution during homologous recombination .

Relevance to Treponema denticola Pathogenicity

Treponema denticola is an oral spirochete that contributes to chronic periodontitis . The dentilisin operon in T. denticola expresses genes encoding proteins like PrcB, PrcA, and PrtP, which are involved in protease activity and interactions within the bacterial cell surface . Although RuvC is not directly part of the dentilisin complex, its role in DNA repair and genetic stability is crucial for the long-term survival and adaptation of T. denticola within the oral environment.

Research Techniques and Findings

Studying RuvC involves various molecular biology and biochemical techniques, such as:

Findings from these techniques contribute to a deeper understanding of RuvC's role in DNA metabolism and bacterial survival.