ABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Uracil-DNA Glycosidase/chemistry , Viral Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Herpesvirus 1, Human/enzymology , Herpesvirus 4, Human/enzymology , Humans , Hydrogen Bonding , Models, Molecular , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Staphylococcus aureus , Uracil-DNA Glycosidase/genetics , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
Bacteriophage T4/metabolism , DNA, Viral/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Fimbriae Proteins/metabolism , Viral Proteins/metabolism , Bacteriophage T4/genetics , DNA Restriction Enzymes/genetics , DNA Restriction Enzymes/metabolism , DNA, Viral/genetics , Escherichia coli/genetics , Escherichia coli/virology , Escherichia coli Proteins/genetics , Fimbriae Proteins/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Viral , Gene Silencing , Protein Binding , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
DNA-Binding Proteins/chemistry , Molecular Mimicry , Animals , Binding Sites , Computational Biology , DNA Repair/drug effects , DNA-Binding Proteins/drug effects , Drosophila Proteins/chemistry , Exodeoxyribonuclease V/antagonists & inhibitors , Histone Acetyltransferases/chemistry , Humans , Models, Molecular , Replication Protein A/chemistry , TATA-Binding Protein Associated Factors , Transcription Factor TFIID/chemistry , Tumor Suppressor Protein p53/chemistry , Uracil-DNA Glycosidase/antagonists & inhibitors , Uracil-DNA Glycosidase/chemistry , Viral Proteins/chemistryABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Models, Molecular , Protein Binding , Staphylococcus epidermidisABSTRACT
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.
