RETRACTED: Structural analysis of BRCA1 reveals modification hotspot

Liang, Yen Nan; Dearnaley, W. J.; Varano, A. Cameron; Winton, Carly E.; Gilmore, Brian L.; Alden, Nick A.; Sheng, Zhi; Kelly, Deborah F.

doi:10.1126/sciadv.1701386

Cited by 17 publications

(24 citation statements)

References 41 publications

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“…Although no high resolution structures of full-length BRCA1 are available, a low resolution negative stain EM structure of the BRCA1-BARD1 complex has been reported, in which it adopts a clamp-like architecture (Fig. 5c) [142].The RING domain and BRCT domain are mapped into the two opposite ends of the map. The presence of additional density near the RING domain is interpreted as ubiquitin in BRCA1 mutants.…”

Section: The Rad51 Mediatorsmentioning

confidence: 99%

Structural basis of homologous recombination

Sun

McCorvie

Yates

et al. 2019

Cell. Mol. Life Sci.

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Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase. Keywords Homologous recombination • Cryo electron microscopy • X-ray crystallography • Double-strand break repair • DNA damage signalling and repair Abbreviations ADP Adenosine diphosphate AMP-PNP Adenylyl-imidodiphosphate ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3-related protein BARD1 BRCA1-associated RING domain protein 1 BRCA1 Breast cancer type 1 susceptibility protein BRCA2 Breast cancer type 2 susceptibility protein BRCT BRCA1 C-terminal domain cryoEM Cryo electron microscopy DDR DNA damage response DSB Double-strand break EJ End joining MRE11 Meiotic recombination 11 homolog 1 MRN MRE11 RAD50 NBS1 NBS1 Nijmegen breakage syndrome protein 1 OB Oligonucleotide/oligosaccharide-binding PALB2 Partner and localiser of BRCA2 RPA Replication protein A

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Section: The Rad51 Mediatorsmentioning

confidence: 99%

Structural basis of homologous recombination

Sun

McCorvie

Yates

et al. 2019

Cell. Mol. Life Sci.

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“…One example of this approach includes our recent studies on breast cancer susceptibility protein (BRCA1)-protein assemblies isolated from breast cancer cells (Figure 1). [6,7] Other successful examples include the high-resolution work of Scheres and colleagues, who determined 3D structures of tau filaments isolated the brains of dementia patients. [8] Hence, engineering new tools to evaluate native protein assemblies is important to advancing the biomedical mission.…”

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confidence: 99%

“…A full description of the biochemical purification scheme used to obtain the BRCA1 complexes was recently published. [7] This protein specimen is an appealing model as its molecular weight and particle diameter (≈300 kDa, 100 Å) is much smaller than that of rotavirus assemblies (≈2.5 MDa, 800 Å). Purified BRCA1 complexes were prepared in aqueous buffer solution containing 50 × 10 −3 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 150 × 10 −3 M NaCl, 10 × 10 −3 M MgCl 2 , 10 × 10 −3 M CaCl 2 .…”

mentioning

confidence: 99%

“…An exciting new direction for the cryo‐EM field involves the structure determination of medically relevant macromolecules for therapeutic design purposes. One example of this approach includes our recent studies on breast cancer susceptibility protein (BRCA1)‐protein assemblies isolated from breast cancer cells ( Figure ) . Other successful examples include the high‐resolution work of Scheres and colleagues, who determined 3D structures of tau filaments isolated the brains of dementia patients .…”

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confidence: 99%

“…Upon removing the microchip from the sample, the excessive buffer solution is blotted onto Whatman filter paper. The microchip is then loaded onto a freezing device and plunged into a slurry of liquid ethane using standard protocols . Optimal specimen preservation conditions on the freezing device include a blotting step of ≈2 s µL −1 of sample volume and a dwell time of ≈1 s.…”

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Cryo‐EM‐On‐a‐Chip: Custom‐Designed Substrates for the 3D Analysis of Macromolecules

Alden

Varano

Dearnaley

et al. 2019

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The fight against human disease requires a multidisciplinary scientific approach. Applying tools from seemingly unrelated areas, such as materials science and molecular biology, researchers can overcome long-standing challenges to improve knowledge of molecular pathologies. Here, custom-designed substrates composed of silicon nitride (SiN) are used to study the 3D attributes of tumor suppressor proteins that function in DNA repair events. New on-chip preparation strategies enable the isolation of native protein complexes from human cancer cells. Combined techniques of cryo-electron microscopy (EM) and molecular modeling reveal a new modified form of the p53 tumor suppressor present in aggressive glioblastoma multiforme cancer cells. Taken together, the findings provide a radical new design for cryo-EM substrates to evaluate the structures of disease-related macromolecules.

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