Affinity For dsDNA Research Articles

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance

Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to γ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans.

Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding

The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in double-stranded DNA (dsDNA). Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and molecular dynamics simulations of homologous homeodomain-DNA complexes. In contrast, our nuclear magnetic resonance (NMR) studies show that DNA binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17-Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a 2-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain-DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17-Arg52 salt bridge in the DNA-bound Gbx1.

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.

Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca2+-dependent Double-stranded DNA Binding Activity

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

Abstract 1963: Novel small molecule inhibitor of the endonuclease function of the APE1 DNA repair and redox signaling enzyme blocks proliferation and reduces viability of glioblastoma cells

Abstract The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP Endonuclease1 (APE1), a critical component in this pathway that hydrolyzes the phosphodiester backbone 5’ to the AP site. Failure to repair AP sites leads to DNA strand breaks, enhanced cytotoxicity, cell cycle arrest and increased apoptosis. In addition to APE1's role in repair, it also functions as a redox signaling factor to activate key transcription factors such as AP1 (Fos/Jun), p53, NFkB, HIF-1α, and others controlling the expression of genes important for cell survival and cancer promotion and progression. APE1 has been shown to have an altered level of expression in a variety of cancers including pancreatic, breast, prostate, gliomas, sarcomas and others. High APE1 expression has also been associated with chemoradiotherapy poor outcome, poor complete response rate, shorter local relapse-free interval, poorer survival and high angiogenesis. Due to APE1's critical role in repairing chemotherapy- and IR-induced DNA damage, we hypothesize that inhibiting APE1's DNA repair activity would not only allow us to further elucidate the role of DNA repair that elevated levels of APE1 play in tumors, but would also allow us to delineate the importance of its DNA repair and redox functions in normal cellular processes. While reducing the amount of APE1 in cancer cells using anti-sense oligonucleotide technology and RNA interference sensitizes tumor cells to chemotherapeutic agents, it may not be the most efficacious route of treatment. We have concluded a screen of small molecule compounds for their ability to inhibit APE1 AP endonuclease activity. Four families of compounds with IC50 values less than 10 μM were identified, validated and characterized. One of the most promising compounds, designated AR03 inhibited cleavage of AP sites in vivo in SF767 glioblastoma cells, in vitro in whole cell extracts and inhibits purified human APE1 in vitro. AR03 has low affinity for dsDNA and it does not affect the redox function of APE1. AR03 also potentiates the cytotoxicity of methyl methane sulfonate (MMS) and temozolomide (TMZ) in SF767 cells. AR03 is chemically distinct from all the previously reported small molecule inhibitors of APE1 which are either ineffective in vivo due to poor cellular uptake, not tested in cellular based scenarios, or lacking in specificity. Thus, AR03 is a novel small molecule inhibitor of APE1, which may have potential as an oncotherapeutic drug for treating glioblastoma and other cancers. Additional studies are needed to determine the effect of AR03 in combination with other cancer chemotherapeutic agents, in combination with other APE1 inhibitors or other emerging BER targets such as PARP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1963.

Different Residues on the Surface of theMethanothermobacter thermautotrophicusMCM Helicase Interact with Single- and Double-Stranded DNA

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM protein, while the N-terminal portion plays an important role in DNA binding and protein multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.

Structure-Function Relationships in Anti-DNA and Anti-Phospholipid Antibodies and their Relevance to the Pathogenesis of Disease

Autoantibodies are pathogenic in systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). These pathogenic autoantibodies are generally characterized by IgG isotype and high affinity binding to particular antigens. In SLE, antibodies to double-stranded DNA (dsDNA), nucleosomes and alpha-actinin are particularly important. In APS, pathogenic antibodies that cause thrombosis or fetal loss are particularly characterized by binding to anionic phospholipids (PL) and beta-2-glycoprotein I. Sequence analysis of human and murine monoclonal anti-dsDNA and aPL antibodies shows that high affinity for these antigens is associated with the presence of the residues arginine (Arg), asparagine (Asn) and lysine (Lys) in the complementarity determining regions (CDRs) of their heavy and light chains. In vitro expression systems have been used to create variants of the antibodies in which these amino acids have been altered. In general, removal of arginine residues reduces affinity for dsDNA, nucleosomes and anionic PL. Arginines at different positions in the sequence have different effects on binding affinity and effects on binding are not always mirrored by effects on pathogenicity. These studies, together with molecular models of antigen/antibody complexes, help us to understand exactly how pathogenic antibodies interact with antigens. Ultimately, this understanding may aid the design of therapeutic agents to block the pathogenic effects of these antibodies.

Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications

BackgroundEvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions.ResultsThis study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our results also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles.ConclusionThese findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications.

Preparation of cationic comb-type copolymer having guanidino moieties and its interaction with DNAs

In order to evaluatet effects of different cationic moieties, such as primary amino and guanidino groups, on interactions between DNAs and cationic comb-type copolymers, comb-type copolymers having guanidino groups were prepared. The copolymers (GPLL-g-Dex) were obtained by guanidination of poly(L-lysine)-graft-dextran copolymers (PLL-g-Dex) using 1-guanyl3,5-dimethylpyrazole nitrate under weak basic conditions. The resulting copolymers were characterized using NMR spectroscopy and size-exclusion chromatography-multiangle light scattering (SECMALS). The primary amino groups of the PLL backbones were thoroughly replaced with guanidino ones without any detectable side reactions, including fragmentation of PLL or Dex chains. The interactions of GPLL-g-Dex and PLL-g-Dex with DNAs were assessed by UV-melting curve measurements. These copolymers diversly affected the melting behavior of double-stranded DNA (dsDNA). GPLL-g-Dex has a lower ability to increase the T m of dsDNA than PLL-g-Dex and also exhibits a higher affinity for dsDNA. The results suggest that the stability of dsDNA may be affected not only by ionic interaction between the copolymers and DNAs, but also by other modes of interactions, such as hydrogen-bondinginteractions.

Characterization of human herpesvirus 8 ORF59 protein (PF-8) and mapping of the processivity and viral DNA polymerase-interacting domains.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

Fluorescence Energy-Transfer Cyanine Heterodimers with High Affinity for Double-Stranded DNA I. Synthesis and Spectroscopic Properties

We have designed, synthesized, and characterized fluorescent cyanine heterodimers that exploit resonance energy transfer to achieve strong emission above 650 nm with 488-nm excitation. Thiazole orange serves as the common fluorescence donor in these dyes and thiazole-indolenine, thiazole blue, or symmetric thiazole blue as accepters. The donor and acceptor chromophores are linked by a polymethylene linker containing quaternary amino groups. These heterodimers have a high affinity for double-stranded DNA (dsDNA). The donor emission in the dsDNA-bound dyes is quenched by over 85%. The affinity for dsDNA and the quenching of donor fluorescence were optimized by varying the length of the linker between the donor and acceptor. Complexes of dsDNA fragments with such optimized dyes dissociated very slowly ( t 0.5 > 300 min) during agarose gel electrophoresis.

Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice

We investigated the capacity of five human monoclonal IgG anti-DNA antibodies derived from lupus patients to produce glomerular immune deposits. The hybridomas secreting these antibodies were administered intraperitoneally to severe combined immunodeficiency (SCID) mice. Three of the five antibodies (B3, 35.21, 33.C9) were detected in the kidneys, but only one (33.C9) deposited exclusively in the glomeruli in the mesangium and capillary wall, whereas the other two antibodies bound to nuclei both in the kidney and in other organs. The antibodies were tested against a variety of autoantigens by ELISA, the only unique feature of 33.C9 was that it also bound strongly to histones. There were no particular amino acid motif that was related to immunoglobulin deposition in the kidney. All the mice that had immunoglobulin deposited in the kidney, either extracellularly or intranuclearly developed 2 to 3+ proteinuria, whereas the other mice had only trace amounts of proteinuria. This study demonstrates that some human monoclonal IgG anti-dsDNA antibodies are capable of binding to the glomerulus while others can penetrate cells and bind to nuclei in vivo. Although no abnormal pathology was observed, proteinuria was detected, perhaps representing an early phase of disease. These results indicate that the affinity for dsDNA is not the sole determining factor governing the biological properties of human anti-DNA antibodies in vivo.

Structural properties of a subset of nephritogenic anti-DNA antibodies.

Structural analysis of lupus autoantibodies is beginning to provide clues to the molecular basis for antigenic specificity and pathogenicity. The present analysis indicates that multiple light and heavy chains contain residues which can facilitate DNA binding, reaffirming the notion that there are multiple ways that different amino acids combine to form an antigen-binding pocket with affinity for dsDNA and ssDNA. Furthermore, this analysis suggests that these conformations and contact residues are intrinsic to germline sequences, although amino acid changes at critical locations (somatically introduced) modulate antigen binding, and appear to influence the capacity of individual immunoglobulin to form immune deposits. Analysis of additional individual immunoglobulins with closely related V-region sequences and differing pathogenic properties will be required to resolve the contribution of specific motifs to pathogenecity.

Site-directed mutagenesis of the yeast PRP20/SRM1 gene reveals distinct activity domains in the protein product.

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.