Association of muscle disorders in late postmenopausal women according to the type of experienced menopause.

OBJECTIVE: Musculoskeletal disorders frequently affect postmenopausal women. This study aims to compare muscle disorders between women according to the type of experienced menopause: premature (PM) or normal age of menopause (NAM). METHODS: This was a cross-sectional study conducted in nine Latin American countries in which late postmenopausal women (55 to 70 years) were surveyed with a general questionnaire, the Menopause Rating Scale (MRS: item #4 exploring musculoskeletal discomfort), and strength, assistance with walking, rising from a chair, climbing stairs, and falling questionnaire (risk of sarcopenia). RESULTS: A total of 644 women were included: 468 who had NAM, and 176 who had PM (116 spontaneous and 60 surgical). The overall mean age of the participants was 60.9 ± 4.2 years. Women who had PM experienced more musculoskeletal discomfort (33.5% vs 20.9%, P < 0.001) and a higher likelihood of sarcopenia (35.2% vs 19.9%, P < 0.001) than women who had a NAM. Women who had surgical PM exhibited a higher prevalence of severe musculoskeletal discomfort (46.7% vs 29.3%, P < 0.02) and a higher likelihood of sarcopenia (45.0% vs 27.6%, P < 0.02) than women who had a NAM. After adjusting for covariates (age, body mass index, menopausal hormone therapy use, physical activity, education, cigarette consumption, use of antidepressants, sexual activity, comorbidities, and having a partner), our logistic regression model determined that spontaneous PM was not associated with higher odds of musculoskeletal discomfort and higher odds of sarcopenia. On the other hand, women who had surgical PM were more likely to experience musculoskeletal discomforts (odds ratio: 2.26; 95% confidence interval: 1.22-4.17) and higher odds for sarcopenia (odds ratio: 2.05; 95% confidence interval: 1.16-3.65) as compared to women who experienced a NAM. CONCLUSIONS: Women experiencing surgical PM have a higher likelihood of developing muscle disorders. This underscores the potential significance of hormonal levels in influencing musculoskeletal health during postmenopause.

Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting.

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+ domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATPγS-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.

Circulating cell-free-DNA concentration is a good biomarker for diagnosis of colorectal cancer in Mexican patients.

Colorectal cancer (CRC) is the third most common cancer in the world. Overall survival is related to clinical stage: more advanced stages show lower survival rates; therefore, they need to be monitored regularly with new, less invasive and more specific biomarkers. The concentration and integrity index of circulating cell-free DNA (ccfDNA) have been proposed as potential diagnostic and prognostic biomarkers for CRC, however, inconsistent results are still observed in different reports. Here we analyze these potential CRC biomarkers in a Mexican population. In this study, 124 patients with sporadic CRC and 37 healthy individuals were examined as a reference group. The ccfDNA was isolated from plasma samples of all included subjects. The ccfDNA concentration was determined by fluorometry and the integrity index (ALU247/ALU115 ratio) by quantitative PCR amplification (qPCR) of ALU sequences. The results show that ccfDNA concentration was higher in CRC patients than in the reference group (P=0.001). The integrity index showed no significant differences between these groups (P=0.258), except for histological type (P=0.012). A higher ccfDNA concentration was also associated with patients younger than 50 years (P=0.030). The ccfDNA concentration showed significant discriminatory power (AUC: 0.854, C.I.: 0.78-0.92, P=0.001) between patients and the reference group and between tumor-node-metastasis (TNM) stages. In conclusion, ccfDNA concentration proves to be a good diagnostic biomarker for CRC patients, whereas the integrity index did not show diagnostic utility.

Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein.

eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

Three-Component Synthesis of 2-Amino-3-cyano-4H-chromenes, In Silico Analysis of Their Pharmacological Profile, and In Vitro Anticancer and Antifungal Testing.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a-o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a-h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a-o and 6a-h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a-o and 6a-h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism.

eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

The transcription factor NFAT5 limits infection-induced type I interferon responses.

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNß as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors.

Synthesis and highly potent anti-inflammatory activity of licofelone- and ketorolac-based 1-arylpyrrolizin-3-ones.

Since NSAIDs are commonly used anti-inflammatory agents that produce adverse effects, there have been ongoing efforts to develop more effective and less toxic compounds. Based on the structure of the anti-inflammatory pyrrolizines licofelone and ketorolac, a series of 1-arylpyrrolizin-3-ones was synthesized. Also prepared was a series of substituted pyrroles, mimicking similar known anti-inflammatory agents. The anti-inflammatory activity of the test compounds was determined with a phorbol ester (TPA)-induced murine ear edema protocol. For the most active derivatives, 19b-c/20b-c, the anti-inflammatory effect was the same as that of the reference compound (indomethacin) and was dose-dependent. These compounds have an aryl ring at the C-1 position and a methoxycarbonyl group at the C-2 position of the pyrrolizine framework, which represent plausible pharmacophore groups with anti-inflammatory activity. The anti-inflammatory activity of 1-substituted analogs containing a five- or six-membered heterocycles was lower but still good, while that of the pyrroles was only moderate. Although the docking studies suggests that the effect of analogs 19a-c/20a-c is associated with the inhibition of cyclooxygenase-2, experimental assays did not corroborate this idea. Indeed, a significant inhibition of NO was found experimentally as a plausible mechanism of action.

Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy.

Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.

UNLABELLED: The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA(+) domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE: The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1.

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains.

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.

Adeno-associated virus Rep represses the human integration site promoter by two pathways that are similar to those required for the regulation of the viral p5 promoter.

UNLABELLED: Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5' untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE: The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.

Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA(+) domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.

The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.