RESUMEN
Uracil-DNA glycosylases (UDGs) are highly conserved proteins that can be found in a wide range of organisms, and are involved in the DNA repair and host defense systems. UDG activity is controlled by various cellular factors, including the uracil-DNA glycosylase inhibitors, which are DNA mimic proteins that prevent the DNA binding sites of UDGs from interacting with their DNA substrate. To date, only three uracil-DNA glycosylase inhibitors, phage UGI, p56, and Staphylococcus aureus SAUGI, have been determined. We show here that SAUGI has differential inhibitory effects on UDGs from human, bacteria, Herpes simplex virus (HSV; human herpesvirus 1) and Epstein-Barr virus (EBV; human herpesvirus 4). Newly determined crystal structures of SAUGI/human UDG and a SAUGI/HSVUDG complex were used to explain the differential binding activities of SAUGI on these two UDGs. Structural-based protein engineering was further used to modulate the inhibitory ability of SAUGI on human UDG and HSVUDG. The results of this work extend our understanding of DNA mimics as well as potentially opening the way for novel therapeutic applications for this kind of protein.
Asunto(s)
Proteínas Bacterianas/química , Uracil-ADN Glicosidasa/química , Proteínas Virales/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 4/enzimología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Staphylococcus aureus , Uracil-ADN Glicosidasa/genética , Proteínas Virales/genéticaRESUMEN
The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties.
Asunto(s)
Bacteriófago T4/metabolismo , ADN Viral/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Proteínas Virales/metabolismo , Bacteriófago T4/genética , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , ADN Viral/genética , Escherichia coli/genética , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , Regulación Viral de la Expresión Génica , Silenciador del Gen , Unión Proteica , Proteínas Virales/genéticaRESUMEN
DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.
Asunto(s)
Proteínas de Unión al ADN/química , Imitación Molecular , Animales , Sitios de Unión , Biología Computacional , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Drosophila/química , Exodesoxirribonucleasa V/antagonistas & inhibidores , Histona Acetiltransferasas/química , Humanos , Modelos Moleculares , Proteína de Replicación A/química , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID/química , Proteína p53 Supresora de Tumor/química , Uracil-ADN Glicosidasa/antagonistas & inhibidores , Uracil-ADN Glicosidasa/química , Proteínas Virales/químicaRESUMEN
The teicoplanin-associated locus regulator (TcaR) regulates gene expression of proteins on the intercellular adhesion (ica) locus involved in staphylococci poly-N-acetylglucosamine biosynthesis. The absence of TcaR increases poly-N-acetylglucosamine production and promotes biofilm formation. Until recently, the mechanism of multiple antibiotic resistance regulator family protein members, such as TcaR, was restricted to binding double-stranded DNA. However, we recently found that TcaR strongly interacts with single-stranded DNA, which is a new role for this family of proteins. In this study, we report Staphylococcus epidermidis TcaR-single-stranded DNA complex structures. Our model suggests that TcaR and single-stranded DNA form a 61-symmetry polymer composed of TcaR dimers with single-stranded DNA that wraps outside the polymer and 12 nt per TcaR dimer. Single-stranded DNA binding to TcaR involves a large conformational change at the DNA binding lobe. Several point mutations involving the single-stranded DNA binding surface validate interactions between single-stranded DNA and TcaR. Our results extend the novel role of multiple antibiotic resistance regulator family proteins in staphylococci.
Asunto(s)
Proteínas Bacterianas/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Unión Proteica , Staphylococcus epidermidisRESUMEN
White spot syndrome virus (WSSV) is a large ( approximately 300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently known DNA mimic proteins.