Recombinant Drosophila grimshawi DNA repair and recombination protein RAD54-like (okr), partial

Basic Research Questions

• What is the functional role of RAD54-like protein (okr) in Drosophila grimshawi?

RAD54-like protein (okr) in Drosophila grimshawi functions primarily in the recombinational DNA repair pathway, where it is involved in both mitotic DNA repair and meiotic recombination. The protein plays an essential role in interhomolog gene conversion (GC), although it may have a less significant role in intersister GC compared to the spn-A/Rad51 protein .

When DNA damage occurs, okr functions in the presence of DNA where spn-A/Rad51 enhances its ATPase activity. This enhancement is crucial for the repair process . Studies in related Drosophila species have demonstrated that RAD54 homologs are critical for female fertility and proper development, suggesting similar importance for okr in D. grimshawi .

• How is the RAD54-like protein (okr) structurally characterized in Drosophila grimshawi?

The RAD54-like protein (okr) in Drosophila grimshawi is a 786 amino acid protein with a molecular mass of approximately 89.2 kDa . As a member of the SNF2/RAD54 helicase family, it contains conserved motifs characteristic of proteins involved in ATP-dependent chromatin remodeling and DNA repair processes .

The protein's sequence reveals several functional domains common to RAD54 family proteins, including regions for ATP binding and hydrolysis, DNA interaction, and protein-protein interactions, particularly with Rad51. The complete amino acid sequence has been determined and can be analyzed for functional motifs through comparison with well-characterized RAD54 proteins from other species .

For structural analysis of okr, researchers typically employ a combination of sequence-based predictions and experimental approaches:

• What experimental approaches are optimal for studying RAD54-like protein (okr) function in Drosophila grimshawi?

Studying the function of RAD54-like protein (okr) in Drosophila grimshawi requires a multi-faceted approach combining genetic, biochemical, and cell biological techniques:

Genetic approaches provide insights into the in vivo function of okr. Random mutagenesis has been successfully used to generate null mutants of the RAD54 homolog in D. melanogaster , and similar approaches could be applied to D. grimshawi. CRISPR-Cas9 gene editing offers more precise manipulation for creating specific mutations in functional domains.

Phenotypic characterization of okr mutants would typically involve assessing DNA damage sensitivity using agents such as X-rays or methyl methanesulfonate (MMS). In D. melanogaster, RAD54-deficient larvae show high sensitivity to these agents . Similarly, fertility studies are crucial, as female sterility is a characteristic phenotype of RAD54 deficiency in Drosophila .

Biochemical approaches are essential for understanding the molecular function of okr:

Advanced imaging techniques, such as immunofluorescence microscopy to track protein localization during DNA repair and single-molecule approaches to observe real-time protein dynamics, provide valuable spatial and temporal information about okr function .

• How conserved is the RAD54-like protein (okr) across Drosophila species and other organisms?

RAD54-like protein shows remarkable conservation across evolutionary lineages, underscoring its fundamental importance in DNA repair mechanisms. The Drosophila melanogaster RAD54 homolog (DmRAD54) displays 46-57% identity to its homologs from yeast and mammals , indicating substantial conservation of this protein even between distantly related organisms.

Although direct comparison data between D. grimshawi okr and other Drosophila RAD54 proteins is not explicitly provided in the available literature, the high conservation between D. melanogaster and more distant species suggests that functional domains and critical residues are likely preserved among Drosophila species .

This evolutionary conservation extends to function as well as sequence. Studies in D. melanogaster have demonstrated that RAD54-deficient flies exhibit phenotypes consistent with defective double-strand break repair, including hypersensitivity to DNA-damaging agents and defects in mitotic recombination . Similar phenotypes are observed in yeast and mammalian systems with RAD54 deficiencies, supporting functional conservation of the recombinational repair pathway throughout evolution .

Advanced Research Questions

• How does the ATPase activity of RAD54-like protein (okr) contribute to its function in DNA repair?

The ATPase activity of RAD54-like protein (okr) is fundamental to its function in DNA repair and recombination. This enzymatic activity provides the energy required for okr to act as a motor protein during critical stages of the homologous recombination process.

In Drosophila grimshawi, as in other organisms, the ATPase activity of okr is enhanced by the presence of DNA and further stimulated by interaction with Rad51 (spn-A in Drosophila) . This enhanced ATPase activity drives several key functions:

First, it powers the translocation of presynaptic complexes (PSCs) along DNA during the homology search phase. Studies in yeast have demonstrated that Rad54 translocates at approximately 170 base pairs per second, and this translocation is essential for efficient homology search .

Second, the ATPase activity enables Rad54 to open up donor double-stranded DNA, creating an underwound DNA bubble-like structure that facilitates homology searching within the DNA . Without this ATP-dependent unwinding activity, the homology search process would be severely impaired.

Third, it drives the postsynaptic phase of recombination by stimulating heteroduplex DNA extension of established joint molecules . Experiments have shown that ATPase-deficient Rad54 mutants (e.g., Rad54-K341R in yeast) show significantly reduced target recognition during homology search .

• What are the key protein-protein interactions of RAD54-like protein (okr) during homologous recombination?

RAD54-like protein (okr) engages in several critical protein-protein interactions that orchestrate the homologous recombination process. The most significant of these interactions is with RAD51 (spn-A in Drosophila), which forms nucleoprotein filaments on single-stranded DNA .

This interaction with RAD51 serves multiple functions. First, RAD51 enhances the ATPase activity of RAD54/okr in the presence of DNA , which is crucial for powering the motor functions of RAD54. Second, RAD54 directly interacts with RAD51 nucleoprotein filaments to enhance synapsis—the homologous pairing with a double-stranded DNA partner . This interaction depends specifically on the N-terminal domain of RAD54, as demonstrated in studies with chimeric proteins .

Research in yeast has revealed that RAD54 can also functionally interact with RDH54, another member of the SNF2/RAD54 family. These proteins occupy distinct binding sites within the presynaptic complex and can act cooperatively during homology search . RDH54 has been shown to stimulate RAD54-driven translocation, with presynaptic complexes containing both proteins translocating 47% faster than those with RAD54 alone .

RAD54 also indirectly interacts with Replication Protein A (RPA), which binds to the DNA bubble created by RAD54 during homology search. Approximately three molecules of RPA bind to this unwound region, stabilizing the opened DNA structure .

• How do null mutations in RAD54-like protein (okr) affect DNA damage sensitivity and development in Drosophila models?

Null mutations in RAD54 homologs have profound effects on both DNA damage sensitivity and development in Drosophila models. Studies in D. melanogaster provide a framework for understanding the likely consequences of okr mutations in D. grimshawi.

DmRAD54-deficient flies exhibit remarkable hypersensitivity to DNA-damaging agents. Larvae lacking functional RAD54 show significantly increased sensitivity to X-rays and methyl methanesulfonate (MMS), indicating defects in double-strand break repair pathways . Additionally, these mutants demonstrate defects in X-ray-induced mitotic recombination, as measured by somatic mutation and recombination tests . These findings confirm the critical role of RAD54 in DNA repair processes.

The developmental requirement for RAD54 appears to be conserved across species, suggesting an evolutionary ancient and essential role in multicellular organisms .

• What is the relationship between RAD54-like protein (okr) expression and developmental stages in Drosophila?

The expression pattern of RAD54-like proteins across developmental stages provides important insights into their biological functions. While specific data for okr expression in D. grimshawi is not extensively documented, studies in the related D. melanogaster offer valuable information that likely applies to other Drosophila species.

The increased expression in early embryos likely reflects the critical need for efficient DNA repair during the rapid nuclear divisions that characterize early Drosophila embryogenesis. During these synchronous nuclear divisions, DNA replication occurs at an exceptionally rapid pace, potentially generating replication-associated DNA damage that requires RAD54-mediated repair.

The elevated expression in ovarian tissue suggests a role in meiotic recombination during oogenesis, as well as potential maternal loading of the protein into eggs to support early embryonic development before zygotic gene expression is fully activated.

• How does RAD54-like protein (okr) contribute to the postsynaptic phase of DNA strand exchange?

RAD54-like protein (okr) plays a pivotal role in the postsynaptic phase of DNA strand exchange during homologous recombination. While specific information for D. grimshawi okr is limited, studies in model organisms, particularly yeast, have elucidated the mechanisms by which RAD54 promotes this critical step in DNA repair.

In the postsynaptic phase, after RAD51 has catalyzed the initial strand invasion and pairing, RAD54 stimulates heteroduplex DNA extension of established joint molecules . This function depends critically on both the ATPase activity of RAD54 and specific protein-protein interactions between RAD54 and RAD51 .

Mechanistically, RAD54 functions as a molecular motor during this process. It uses energy from ATP hydrolysis to drive translocation along the DNA, which generates torsional stress in the DNA molecule . This torsional stress helps to extend the heteroduplex region and stabilize the D-loop structure. Studies using DNA curtain assays have demonstrated that yeast Rad54 translocates at approximately 170 base pairs per second and can cover distances of several thousand base pairs .

RAD54 also plays a role in removing RAD51 from heteroduplex DNA after strand invasion, which is necessary for subsequent steps in recombination, including DNA synthesis by polymerases. This removal function is also dependent on the ATPase activity of RAD54.

• What experimental methods can be used to assess RAD54-like protein (okr) translocation on DNA?

Assessing the translocation activity of RAD54-like protein (okr) on DNA requires sophisticated experimental approaches that can detect protein movement along DNA molecules. Several methodologies have been developed for this purpose, each with distinct advantages and limitations.

DNA curtain assays represent one of the most powerful approaches for directly visualizing protein translocation on DNA . In this single-molecule technique, DNA molecules are anchored to a lipid bilayer on a microfluidic surface and stretched by buffer flow. Fluorescently labeled proteins can then be observed as they move along these "curtains" of DNA molecules. This approach has been successfully used to demonstrate that yeast Rad54 translocates at approximately 170 base pairs per second over distances of several thousand base pairs . When combined with Rdh54, the translocation rate increases to about 250 base pairs per second .

Triplex displacement assays provide an alternative approach for measuring translocation in a bulk biochemical format. In this method, a triplex-forming oligonucleotide is bound to a specific site on a DNA molecule. As RAD54 translocates along the DNA, it displaces the triplex, which can be detected through changes in fluorescence or by gel electrophoresis.

• How do RAD54-like protein (okr) and RAD51 cooperate during homologous recombination?

The cooperation between RAD54-like protein (okr) and RAD51 (spn-A in Drosophila) represents a sophisticated molecular partnership that orchestrates the complex process of homologous recombination. Their interaction occurs at multiple stages of the recombination process and involves both physical association and functional synergy.

In the presynaptic phase, RAD51 forms nucleoprotein filaments on single-stranded DNA created at sites of DNA damage . During this phase, RAD54 may enhance the stability and activity of these filaments. Importantly, in the presence of DNA, RAD51 enhances the ATPase activity of RAD54/okr , establishing a reciprocal functional relationship.

The synaptic phase sees the most critical cooperation between these proteins. RAD54 directly interacts with RAD51 nucleoprotein filaments to enhance synapsis—the homologous pairing with a double-stranded DNA partner . This enhancement depends on the N-terminal domain of RAD54, which makes specific contacts with RAD51 . DNA curtain assays have demonstrated that presynaptic complexes containing both RAD51 and RAD54 efficiently bind to donor DNA and undergo translocation during homology search .

In the postsynaptic phase, RAD54 stimulates heteroduplex DNA extension of established joint molecules in RAD51/RPA-mediated DNA strand exchange . This stimulation depends both on the ATPase activity of RAD54 and on specific protein-protein interactions between RAD54 and RAD51 .

• How can CRISPR-Cas9 be utilized to study RAD54-like protein (okr) function in Drosophila grimshawi?

CRISPR-Cas9 genome editing technology offers powerful approaches for investigating RAD54-like protein (okr) function in Drosophila grimshawi. This versatile system enables precise genetic manipulations that were previously challenging or impossible with traditional methods.

For fundamental functional analysis, creating null mutations through CRISPR-Cas9 provides a clean genetic background to assess the consequences of complete okr loss. This can be achieved by designing guide RNAs targeting critical exons of the okr gene, resulting in frameshift mutations that produce truncated, non-functional proteins. Based on studies in D. melanogaster, researchers should anticipate that okr null mutants might be viable but female-sterile .

Domain-specific mutations offer more nuanced insights into okr function. The ATPase domain is particularly important, as it powers the motor function of the protein . By introducing specific point mutations in the ATPase domain (e.g., equivalent to the K341R mutation in yeast Rad54), researchers can create catalytically inactive versions of okr while preserving protein structure and interactions.

Tagged versions of okr can be generated by incorporating sequences encoding fluorescent proteins or epitope tags. C-terminal tagging is often preferred as it tends to be less disruptive to protein function. These tagged versions enable protein localization studies, chromatin immunoprecipitation, and protein complex purification.

• What are the comparative functions of RAD54-like protein (okr) and its homologs across different species?

RAD54 proteins demonstrate remarkable functional conservation across evolutionary lineages while also exhibiting species-specific adaptations. This comparative perspective provides valuable insights into both the fundamental and specialized roles of these proteins in DNA repair processes.

In the yeast Saccharomyces cerevisiae, RAD54 plays a crucial role in the recombinational repair pathway . It stimulates the postsynaptic phase of RAD51-mediated DNA strand exchange, and this function depends on both its ATPase activity and specific protein-protein interactions with RAD51 . Yeast RAD54 works in concert with RDH54, another member of the SNF2/RAD54 family with partially overlapping functions . These proteins occupy distinct binding sites within the presynaptic complex and can act cooperatively during homology search .

In Drosophila melanogaster, DmRAD54 is essential for female fertility and early embryonic development . Eggs from DmRAD54-deficient females do not hatch, indicating a critical role in early development . DmRAD54 is also required for efficient repair of double-strand breaks, as evidenced by the hypersensitivity of mutant larvae to X-rays and methyl methanesulfonate . Additionally, DmRAD54 is necessary for X-ray-induced mitotic recombination .

For Drosophila grimshawi okr, available information indicates roles in mitotic DNA repair and meiotic recombination, with particular importance in interhomolog gene conversion . The functional similarity to DmRAD54 suggests evolutionary conservation of its role in DNA repair processes.

• What biochemical assays are suitable for studying the DNA unwinding activity of RAD54-like protein (okr)?

Although RAD54-like protein (okr) belongs to the SNF2/RAD54 helicase family , it functions not as a conventional helicase that completely separates DNA strands, but rather creates transient, localized unwinding during DNA translocation. Several biochemical assays can assess this specialized DNA unwinding activity.

Topological assays measure RAD54's ability to introduce negative supercoiling into circular DNA molecules, which reflects its DNA unwinding activity. As RAD54 translocates along DNA, it can generate negative supercoils ahead of the translocation direction and positive supercoils behind it. These topological changes can be visualized using gel electrophoresis after removal of proteins, with different topoisomers migrating at different rates.

DNA bubble detection assays capture the transient unwinding activity of RAD54. Studies have shown that RAD54 opens a bubble in donor dsDNA during homology search that is bound by approximately three molecules of RPA . This unwinding can be detected using chemical probes that preferentially modify single-stranded DNA or by measuring the binding of single-strand DNA binding proteins like RPA.

D-loop formation assays provide a functional readout of RAD54's unwinding activity in the context of homologous recombination. By assessing the efficiency with which RAD54 stimulates RAD51-mediated D-loop formation, researchers can indirectly measure the protein's ability to unwind donor DNA to facilitate strand invasion.

• How can single-molecule techniques advance our understanding of RAD54-like protein (okr) function?

Single-molecule techniques have revolutionized our understanding of DNA repair proteins, including RAD54, by allowing direct observation of molecular events that are masked in bulk biochemical assays. These approaches offer unique insights into the dynamics, heterogeneity, and mechanistic details of okr function.

DNA curtain assays have been particularly informative for studying RAD54 translocation during homologous recombination . In this technique, DNA molecules are anchored to a lipid bilayer on a microfluidic surface and stretched by buffer flow. Using this approach, researchers have determined that yeast Rad54 translocates at approximately 170 base pairs per second over distances of several thousand base pairs . Furthermore, these assays revealed that when Rad54 and Rdh54 work together, translocation velocity increases by 47% to about 250 base pairs per second .

Single-molecule FRET (Fluorescence Resonance Energy Transfer) can provide detailed information about conformational changes in DNA and protein complexes during RAD54-mediated processes. By strategically placing fluorophores on DNA substrates or on the protein itself, researchers can detect nanometer-scale distance changes that occur during DNA unwinding, protein binding, or complex formation.

Optical or magnetic tweezers allow manipulation of single DNA molecules while measuring forces and torques generated by RAD54 during its motor activity. These techniques can directly measure the mechanical work performed by RAD54 during translocation and unwinding.