Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.     

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.     

HERITABILITY OF AEROBIC POWER OF INDIVIDUALS IN NORTHEAST BRAZIL

The objective of this study was to evaluate the genetic and environmental contribution to variation in aerobic power in monozygotic (MZ) and dizygotic (DZ) twins. The sample consisted of 20 MZ individuals (12 females and 8 males) and 16 DZ individuals (12 females and 4 males), aged from 8 to 26 years, residents in Natal, Rio Grande do Norte. The twins were assessed by a multistage fitness test. The rate of heritability found for aerobic power was 77%. Based on the results, the estimated heritability was largely responsible for the differences in aerobic power. This implies that such measures are under strong genetic influence.     

Nailfold videocapillaroscopy micro-haemorrhage and giant capillary counting as an accurate approach for a steady state definition of disease activity in systemic sclerosis

Introduction

Nailfold videocapillaroscopy (NVC) in systemic sclerosis (SSc) is a procedure commonly used for patient classification and subsetting, but not to define disease activity (DA). This study aimed to evaluate whether the number of micro-haemorrhages (MHE), micro-thrombosis (MT), giant capillaries (GC), and normal/dilated capillaries (Cs) in NVC could predict DA in SSc.

Methods

Eight-finger NVC was performed in 107 patients with SSc, and the total number of MHE/MT, GC, and the mean number of Cs were counted and defined as number of micro-haemorrhages (NEMO), GC and Cs scores, respectively. The European Scleroderma Study Group (ESSG) index constituted the gold standard for DA assessment, and scores ≥3.5 and =3 were considered indicative of high and moderate activity, respectively.

Results

NEMO and GC scores were positively correlated with ESSG index (R = 0.65, P <0.0001, and R = 0.47, P <0.0001, respectively), whilst Cs score showed a negative correlation with that DA index (R = −0.30, P <0.001). The area under the curve (AUC) of receiver operating characteristic plots, obtained by NEMO score sensitivity and specificity values in classifying patients with ESSG index ≥3.5, was significantly higher than the corresponding AUC derived from either GC or Cs scores (P <0.03 and P <0.0006, respectively). A modified score, defined by the presence of a given number of MHE/MT and GC, had a good performance in classifying active patients (ESSG index ≥3, sensitivity 95.1%, specificity 84.8%, accuracy 88.7%).

Conclusions

MHE/MT and GC appear to be good indicators of DA in SSc, and enhances the role of NVC as an easy technique to identify active patients.     

Outcome of Tuberculosis Treatment in Patients with Diabetes Mellitus Treated in the Revised National Tuberculosis Control Programme in Malappuram District,Kerala, India

Settings

Kerala State, India has reported the greatest dual burden of Tuberculosis (TB) and Diabetes Mellitus (DM). Malappuram district in Kerala has monitored and recorded DM status and its control from 2010 under Revised National Tuberculosis Control Program (RNTCP).

Objectives

To assess, under programme conditions, comprehensiveness of recording DM status among TB cases and the TB treatment outcomes among DM patients (disaggregated by glycemic control) and compare with-non DM patients.

Design

This retrospective record review included 3,116TB patients from April 2010 to September 2011.DM was defined as per international guidelines and TB treatment outcomes were categorized as favourable(cured and treatment completed) and unfavourable(death, default, failure and transfer out). Relative Risk (RR) and 95% confidence intervals(CI) were calculated to assess the risk of unfavourable outcomes.

Results

DM status was recorded in 90% of TB cases and 667 (24%) had DM. 17% of DM patients and 23% of patients with unknown DM status had unfavourable outcomes but this difference was not statistically significant. Unadjusted RR for poor glycemic control or unknown control status for unfavourable outcome were (2.00; 95% CI 0.97–4.13) and (2.14; 95% CI 1.11–4.13).

Conclusion

This study could not confirm an adverse association between DM or its control during treatment and the course of response to TB treatment.DM screening in TB cases and recording of DM care needs to be improved to enable more conclusive evidence.     

The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding

Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70–protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70_1–326). Correlation analysis of the amide ¹H and ¹⁵N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70_1–326 with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70_1–326, indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70_1–326 and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70_1–168). We also examined the structure of hRPA70_1–326 after addition of three different ssDNA substrates. Each of these substrates induced specific amide ¹H and/or ¹⁵N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide ¹H and ¹⁵N chemical shift changes of hRPA70_1–168 after addition of ssDNA. The same amide ¹H and ¹⁵N chemical shift changes were observed for the NTD in hRPA70_1–168 and hRPA70_1–326. The NTD residues with the largest amide ¹H and/or ¹⁵N chemical shift changes were localized to a basic cleft that is important for hRPA70–protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70–protein interactions.     

Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1–326 (hRPA70_1–326), and a fragment of the human XPA protein, residues 98–219 (XPA-MBD). Intensity changes were observed for amide resonances in the ¹H–¹⁵N correlation spectrum of uniformly ¹⁵N-labeled hRPA70_1–326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70_1–326 fragment. The hRPA70_1–326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70_1–326 suggests that a competitive binding mechanism mediates the formation of the RPA–XPA complex. To determine whether a ternary complex could form between hRPA70_1–326, XPA-MBD and ssDNA, a ¹H–¹⁵N correlation spectrum was acquired for uniformly ¹⁵N-labeled hRPA70_1–326 after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70_1–326 in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70_1–326 suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70_1–326 may be modulated by ssDNA.     

1H, 15N and 13C resonance assignments for the three LOTUS RNA binding domains of Tudor domain-containing protein TDRD7

The LOTUS or OST-HTH domain is a recently discovered motif of about 80 amino acids and is found in several germline-specific proteins including the Tudor domain-containing proteins TDRD5 and TDRD7, which are important for germ cell development. The LOTUS domain is an RNA binding domain but its exact function is unknown. Here, we report the ¹H, ¹³C and ¹⁵N resonance assignments for the three LOTUS domains present in mouse TDRD7. These assignments will allow three-dimensional structure determination of the LOTUS domains and mapping of their interaction with RNA, steps toward deciphering the function of TDRD7.