A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

Download Full-text

Yeast Nucleolin Nsr1 Impedes Replication and Elevates Genome Instability at an Actively Transcribed Guanine-Rich G4 DNA-Forming Sequence

Genetics ◽

10.1534/genetics.120.303736 ◽

2020 ◽

Vol 216 (4) ◽

pp. 1023-1037

Author(s):

Shivani Singh ◽

Alexandra Berroyer ◽

Minseon Kim ◽

Nayun Kim

Keyword(s):

Dna Binding ◽

Genome Stability ◽

Genome Instability ◽

Functional Characterization ◽

Yeast Genome ◽

Sequence Motifs ◽

G4 Dna ◽

C Terminus ◽

Dna Strand

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin (NCL) is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that the yeast nucleolin Nsr1 is enriched at a G4 DNA-forming sequence in vivo and is a major factor in inducing the genome instability associated with the cotranscriptionally formed G4 DNA in the yeast genome. We also show that Nsr1 results in impeding replication past such a G4 DNA-forming sequence. The G4-associated genome instability and the G4 DNA-binding in vivo require the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of the RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vivo and restores unhindered replication, overall resulting in a sharp reduction in the genome instability associated with a guanine-rich G4 DNA-forming sequence. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and impeding replication.

Download Full-text

Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.678.18 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

Amy E. Rumora ◽

Jon E. Ramsey ◽

Bryan A. Ballif ◽

Robert J. Kelm

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Functional Characterization ◽

Binding Domain ◽

Dna Binding Domain ◽

Single Stranded Dna ◽

The Core ◽

Element Binding Protein ◽

Protein B

Download Full-text

Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5811 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5811-5820 ◽

Cited By ~ 188

Author(s):

T Mikita ◽

D Campbell ◽

P Wu ◽

K Williamson ◽

U Schindler

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Functional Characterization ◽

Sh2 Domain ◽

Binding Domain ◽

Interleukin 4 ◽

Dna Binding Domain ◽

Wild Type ◽

293 Cells ◽

Phosphorylated Peptides

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

Download Full-text

Effect of a salt-bridge between inter-repeats on the 3D structure of the c-Myb DNA-binding domain revealed by thermodynamic analysis

Journal of Thermal Analysis and Calorimetry ◽

10.1007/s10973-017-6524-9 ◽

2017 ◽

Vol 131 (1) ◽

pp. 335-341 ◽

Cited By ~ 4

Author(s):

Satomi Inaba ◽

Harumi Fukada ◽

Masayuki Oda

Keyword(s):

Dna Binding ◽

Thermodynamic Analysis ◽

3D Structure ◽

Salt Bridge ◽

Binding Domain ◽

Dna Binding Domain

Download Full-text

Novel RNA and DNA strand exchange activity of the PALB2 DNA binding domain and its critical role for DNA repair in cells

eLife ◽

10.7554/elife.44063 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Jaigeeth Deveryshetty ◽

Thibaut Peterlini ◽

Mikhail Ryzhikov ◽

Nadine Brahiti ◽

Graham Dellaire ◽

...

Keyword(s):

Breast Cancer ◽

Dna Binding ◽

Critical Role ◽

Binding Domain ◽

Strand Exchange ◽

Dna Binding Domain ◽

Brca1 And Brca2 ◽

Exchange Activity ◽

In Cells

BReast Cancer Associated proteins 1 and 2 (BRCA1, −2) and Partner and Localizer of BRCA2 (PALB2) protein are tumour suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. PALB2 coordinates functions of BRCA1 and BRCA2 during homology-directed repair (HDR) and interacts with several chromatin proteins. In addition to protein scaffold function, PALB2 binds DNA. The functional role of this interaction is poorly understood. We identified a major DNA-binding site of PALB2, mutations in which reduce RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the function of RAD51 recombinase. Surprisingly, it possesses the strand exchange activity without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

Download Full-text