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At least 19 recordsLinked to original sources
PubMed2025

Moss BRCA2 lacking the canonical DNA-binding domain promotes homologous recombination and binds to DNA.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2's functional diversity across species.

BRCA2 Protein↗
PubMed1979

Messenger RNA for the Ad2 DNA binding protein: DNA sequences encoding the first leader and heterogenity at the mRNA 5' end.

During the early stage of Ad2 infection of human cells, RNA is transcribed from five separate transcription units. Early region II encodes the mRNA for a 72K single-stranded DNA binding protein (DBP) which functions in DNA replication. This report describes the structure of the first leader of the DBP mRNA and the flanking sequences in the DNA. The leader, labeled in vivo with 32P, was isolated by DNA filter hybridization to the viral restriction fragment Eco RI F, and its RNAase T1 and RNAase A oligonucleotides were analyzed by RNA fingerprinting techniques. Comparison of this RNA sequence information with the DNA sequence of Eco RI F has located a 68 nucleotide region of the Hae III C subfragment at coordinate 75.1 that encodes the leader. This position is near the coordinate to which nascent chain analysis and ultraviolet transcription mapping have mapped an RNA initiation site, or promoter, for the DBP mRNA. The DNA sequence that overlaps the leader on the 3' side contains a donor sequence for splicing this leader to a second downstream leader. The splicing sequence shows a seven base homology with the comparable structure of the Ad2 major late leader, and a mouse globin mRNA splicing sequence. The DNA sequence upstream from the cap, the region oof the potential promoter site does not, however, contain a "TA-TAAA"-type homology of the sort noted by D. Hogness, M. Goldberg and R. Lifton (personal communication) for many cellular transcription units, and by other investigations for the Ad2 major late transcription unit. Also, the leader is found with two distinct capped 5' termini, 7meGpppA and 7meGpppG, which are encoded at adjacent positions in the DNA and thus are from mRNAs which are staggered by one nucleotide in length at the 5' end. The staggering at the 5' terminus and the lack of the upstream homolgy distinguish the DBP mRNA from many viral and cellular messenger. In both these respects, however, the DBP mRNA resembles the late messengers of SV40 and polyoma viruses. In this paper, we discuss the implications of these findings for the mechanism of specifying mRNA 5' ends.

Adenoviruses, Human↗
PubMed2025

Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

BRCA2 Protein↗
PubMed2026

Activity of natural single nucleotide variants of human alkyladenine DNA glycosylase AAG R145H, G163S and R197C involved in DNA binding.

Alkyladenine DNA glycosylase (AAG) is a critical enzyme in the base excision repair (BER) pathway that safeguards genome integrity by removing structurally diverse alkylated and deaminated purine lesions from DNA. It serves as a primary defense against alkylation-induced mutations, which are linked to cancer development, chronic inflammation, and neurodegenerative diseases. Single nucleotide variants (SNVs) in the gene coding region have the potential to alter the enzyme's functionality, potentially modulating the repair capacity and affecting response and prognosis following chemoradiotherapy. In our study, we investigated three SNVs that lead to amino acid class changes in regions involved in DNA substrate coordination: R145H, G163S, and R197C using an in vitro approach. Using biochemical assays and molecular dynamics simulations, we evaluated the thermal stability, DNA binding affinity, and glycosylase activity of AAG variants toward hypoxanthine (Hx) and 1,N6-ethenoadenosine (εA) containing substrates. The G163S variant showed reduced thermal stability due to the conformational strain in the β-hairpin loop that intercalates in DNA, but retained εA excision activity comparable to that of wild-type AAG, while losing activity against Hx-containing DNA. The R197C variant had a four-fold reduction in DNA binding affinity for both substrates, and was catalytically inactive, unable to excise either damaged bases. This loss of function correlated with the rearrangement of the 201-210 loop and the reorientation of Arg-201 and Arg-207, which disrupts critical DNA contacts. However, the R145H variant retained near-wild-type thermal stability and activity on both substrates, despite bioinformatic predictions of deleterious effect. Molecular dynamics simulations revealed variant-specific structural disruptions. The data obtained underscore the importance of experimental validation in assessing the functional impact SNVs.

DNA Glycosylases↗
PubMed1979

Gene D5 product of bacteriophage T5: DNA-binding protein affecting DNA replication and late gene expression.

Gene D5 is not only necessary for replication of bacteriophage T5 DNA and for shutoff of expression of some early genes, but has been found to be necessary also for the expression of late T5 genes. The polypeptide product of gene D5 has been identified, an intragenic map of gene D5 has been constructed, and the direction of transcription of gene D5 has been established. The polypeptide coded by gene D5 has been shown to be a DNA-binding protein with affinity for both double- and single-stranded DNA.

Coliphages↗
PubMed1979

Malignancy-associated DNA-binding protein C3DP from human serum. Further characterization and purification of C3DP retaining its DNA-binding affinity.

C3DP, a malignancy-associated DNA-binding protein from human serum[1], was purified to homogeneity without loss of its DNA-binding affinity. For this purpose normal human serum was submitted to affinity chromatography on Con A-Sepharose and DNA-cellulose and to preparative polyacrylamide gel electrophoresis. The purified C3DP was identified by immunodiffusion and sodium dodecylsulfate polyacrylamide gel electrophoresis and it was shown to bind to DNA by DNA-cellulose chromatography. The isoelectric point of C3DP was determined to 4.9 by isoelectric focusing.

Chromatography, Affinity↗
PubMed2025

iNOME-seq: in vivo simultaneous genome-wide mapping of chromatin accessibility, nucleosome positioning, DNA-binding protein sites, and DNA methylation in Arabidopsis.

We present iNOMe-seq, a novel method for in vivo simultaneous profiling of chromatin accessibility, nucleosome occupancy, DNA-binding protein sites, and DNA methylation in living tissues. iNOMe-seq utilizes an m5C methyltransferase to mark accessible cytosines in a GpC context, bypassing nucleosome-restricted regions. Using Arabidopsis thaliana, we demonstrate that iNOMe-seq improves chromatin accessibility quantification compared to existing methods. Furthermore, it allows for the spatial and temporal analysis of chromatin dynamics, transcription factor binding, and DNA methylation, offering insight into the role of epigenetic components in transcriptional regulation across tissues and genetic variations in natural populations.

Arabidopsis↗
PubMed1978

Fluorescence and chemical studies on the interaction of Escherichia coli DNA-binding protein with single-stranded DNA.

Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod.

Coliphages↗
PubMed2026

Overlapping RAD18- and DNA-binding interfaces in DNA polymerase η contribute to UV-induced DNA damage tolerance.

DNA polymerase η (Polη) bypasses UV-induced pyrimidine dimers and thereby confers tolerance to UV irradiation. Although the C-terminus of Polη has been reported to interact with ubiquitinated PCNA and RAD18, how Polη engages RAD18 is not fully understood. Here, we show that Polη and RAD18 interact through two distinct modes in human cells: a ubiquitinated-PCNA-dependent mode that requires the Polη C-terminus, and an unexpected PCNA-independent mode mediated by its N-terminal region. We focused our subsequent analyses on this newly identified PCNA-independent mode. Using purified recombinant proteins, we demonstrate direct binding of the N-terminal region of human Polη (PolηΔC) to RAD18 in vitro. Although PolηΔC and RAD18 each bound primer-template DNA, we were unable to detect a ternary PolηΔC-RAD18-DNA complex, and DNA competitively inhibited RAD18 binding to both PolηΔC and full-length Polη. Mutational analyses revealed that the DNA-binding and RAD18-binding domains within Polη overlap. A separation-of-function mutant, PolηΔC(K317A), which retains near-normal DNA-binding and polymerase activities but exhibits reduced RAD18 binding in vitro, displayed a diminished ability to rescue the UV sensitivity of Polη-deficient cells. Notably, the detrimental impact of the K317A persisted in a PCNA-binding-defective background but was attenuated in RAD18-knockout cells. These findings demonstrate that RAD18 binding to the N-terminal domain of Polη contributes to efficient bypass of pyrimidine dimers independently of the Polη-PCNA interaction and provide mechanistic insights into how Polη-RAD18 complexes assemble and dissociate during translesion DNA synthesis.

Journal Article↗
PubMed1976

polA6, A mutation affecting the DNA binding capacity of DNA polymerase I.

The polA6 mutation is an allele of the polA gene of Escherichia coli which produces a DNA polymerase I species readily distinguishable from that produced by the wild type allele. Experiments described here show that this enzyme has an altered pH optimum for polymerization and a lower binding affinity for DNA. The defect clearly lies within the carboxyl-terminal large fragment of the enzyme produced by in vivo or in vitro proteolysis since the fragment has the same pH optimum for polymerization as the intact enzyme. The polA6 enzyme and its fragment are more sensitive to phosphate ions than the wild type polymerase, and the large fragment is less efficient at binding poly d(AT) in in vitro binding assays. Although the specific nucleolytic activity of the polA6 enzyme is higher than that of the wild type, there is no apparent alteration in pH optimum for the hydrolysis of eigher double or single stranded DNA.

Alleles↗
PubMed1979

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).

Carrier Proteins↗
PubMed1979

Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site.

Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.

DNA, Bacterial↗
PubMed1979

Nonrandom correlation between selective distribution of virogenic 5-bromodeoxyuridine and DNA-binding nonhistone proteins in rat DNA.

In order to more accurately determine a mechanism for the bromodeoxyuridine (BrdU)-mediated activation of endogenous type C virus from normal rat embryo cells, nonhistone nuclear protein-DNA interactions were analyzed in vitro. Native, as well as kinetically fractioned DNA samples previously labeled with either [3H]-thymidine or [3H]-BrdU were combined with DNA-binding nonhistones and characterized according to distribution of the isotopes, and extent and localization of protein-binding sites. As before, [3H]-BrdU was relatively more concentrated in repetitive DNA as compared to [3H]-thymidine. Using a membrane filter retention assay, nearly 60% of complete, 37% of repetitive, and 12% of nonrepeated DNA-protein reconstituted complexes were retained on the filters regardless of isotopic precursor. However, a proportionately greater amount of [3H]-BrdU than [3H]-thymidine was recovered following extensive digestion of renatured complexes with DNase I, even though comparable amounts of DNA were acid-insoluble. The disproportionate binding of nonhistones to repetitive DNA, especially BrdU-substituted regions, may be related to the highly specific, well-characterized modifications in eukaryotic transcription attributed to the analog.

Animals↗
PubMed1978

Analysis of DNA structure by hydroxyapatite columns and ethidium bromide fluorescence techniques. A comparative study and effect on DNA binding.

Seven duplex DNA preparations have been structurally analyzed by hydroxyapatite column chromatography and an ethidium bromide fluorescence technique. Significant contamination of one preparation with single-stranded DNA was detected by hydroxyapatite column chromatography. Five of the other six preparations were found to contain significant single-stranded regions by the ethidium bromide fluorescence technique. Synthetic poly dAT was found to be duplex in structure. The presence of single-stranded regions considerably influenced DNA binding results in a radioimmunoassay.

Antibodies↗
PubMed1977

DNA binding and its relationship to carcinogenesis by different polycyclic hydrocarbons.

Five different polycyclic hydrocarbons with different degrees of carcinogenicity in vivo were tested for their metabolism to water-soluble products and their binding to DNA, RNA, and protein in normal embryonic hamster and BHK cells. The compounds studied were 7, 12-dimethylbenz(a)anthracene, benzo(a)pyrene, 20-methyl-cholanthrene, dibenz(a,h)anthracene and dibenz(a,c)anthracene. All five compounds were metabolized to water-soluble produces in both types of cells and treatment of cells with aminophylline enhanced this metabolism. After and not before this enhancement of metabolism by aminophylline, there was a relationship between the degree of carcinogenicity and binding to DNA. There was no such relationship with binding to RNA or protein. The results, indicating a relationship between the degree of carcinogenicity and binding to DNA under appropriate conditions of metabolism, support the suggestion that DNA is the target for carcinogenesis by such carcinogens.

Aminophylline↗
PubMed1977

Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149.

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.

Adenoviruses, Human↗