A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

Clinical relevance and prognostic role of preoperative cell-free single-stranded DNA concentrations in colorectal cancer patients

Purpose: Circulating cell-free single-stranded DNA (ccf-ssDNA) is extracellular DNA and it is a useful biomarker for the diagnosis of tumors and predicting the prognosis of tumors. However, the clinical usefulness of ccf-ssDNA in colorectal cancer (CRC) is not well known. Thus, the purpose of this study was to investigate the clinical usefulness of ccf-ssDNA in CRC.Methods: The study was conducted on 44 patients who had undergone surgery for CRC, and ccf-ssDNA level was measured before surgery and statistical analysis was performed on clinical factors.Results: The association between ccf-ssDNA level and clinicopathological factors was analyzed and compared, and these factors included age, sex, body mass index, diabetes mellitus, hypertension, tumor markers (carcinoembryonic antigen and carbohydrate antigen 19-9), tumor location, size, stage (TNM), recurrence, and death. The group with a ccf-ssDNA level of ≥ 7.5 ng/μL had a lower age (P = 0.010), and was associated with diabetes mellitus (P = 0.037) and lymph node metastasis (P = 0.049). Multivariate analysis of disease-free survival showed that lymph node metastasis and ccf-ssDNA level (hazard ratio, 10.011; 95% confidence interval, 2.269–44.175; P = 0.002) were independent prognostic factors for recurrence. In terms of overall survival, there were no statistically significant results except for vascular invasion.Conclusion: This study showed that ccf-ssDNA level in plasma in CRC patients was an independent prognostic factor that could predict recurrence non-invasively. In this regard, further evaluation with a prospective, large sample size study will be needed to obtain additional results.

A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks

After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3′ single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept.

Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-α/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 Å resolution. The structure reveals a CST and a pol-α/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-α/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-α/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-α/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.

Evaluation of Polytyramine Film and 6-Mercaptohexanol Self-Assembled Monolayers as the Immobilization Layers for a Capacitive DNA Sensor Chip: A Comparison

The performance of a biosensor is associated with the properties of an immobilization layer on a sensor chip. In this study, gold sensor chips were modified with two different immobilization layers, polytyramine film and 6-mercaptohexanol self-assembled monolayer. The physical, electrochemical and analytical properties of polytyramine film and mercaptohexanol self-assembled monolayer modified gold sensor chips were studied and compared. The study was conducted using atomic force microscopy, cyclic voltammetry and a capacitive DNA-sensor system (CapSenze™ Biosystem). The results obtained by atomic force microscopy and cyclic voltammetry indicate that polytyramine film on the sensor chip surface possesses better insulating properties and provides more spaces for the immobilization of the capture probe than a mercaptohexanol self-assembled monolayer. A capacitive DNA sensor hosting a polytyramine single-stranded DNA-modified sensor chip displayed higher sensitivity and larger signal amplitude than that of a mercaptohexanol single-stranded DNA-modified sensor chip. The linearity responses for polytyramine single-stranded DNA- and mercaptohexanol single-stranded DNA-modified sensor chips were obtained at log concentration ranges, equivalent to 10−12 to 10−8 M and 10−10 to 10−8 M, with detection limits of 4.0 × 10−13 M and 7.0 × 10−11 M of target complementary single-stranded DNA, respectively. Mercaptohexanol single-stranded DNA- and polytyramine single-stranded DNA-modified sensor chips exhibited a notable selectivity at an elevated hybridization temperature of 50 °C, albeit the signal amplitudes due to the hybridization of the target complementary single-stranded DNA were reduced by almost 20% and less than 5%, respectively.