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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA
Evan R Guggenheim1, Dong Xu, Christiana X Zhang
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind the DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25 bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand crosslink were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photocrosslinked and identified in cervical, testicular, pancreatic and bone cancer-cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA-PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photocrosslinked only by the probe containing a 1,3-d(G*pTpG*) intrastrand crosslink. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photocrosslinked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photocrosslinking experiments with this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early steps of the mechanism of action of the cisplatin and its close analogue carboplatin than previously was available.
Insights
Researchers identified key nuclear proteins interacting with cisplatin-damaged DNA, advancing our understanding of DNA repair and cancer drug mechanisms. This study reveals more proteins involved in the early action of cisplatin and carboplatin.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Cisplatin's anticancer activity stems from its DNA binding, forming adducts that distort DNA structure.
- These DNA distortions trigger cellular responses, including DNA repair or apoptosis, depending on repair success.
- Understanding protein interactions with platinum-DNA adducts is crucial for elucidating cisplatin's mechanism of action.
Purpose of the Study:
- To identify nuclear proteins that bind to specific platinum-induced DNA adducts formed by cisplatin.
- To investigate the differential binding affinities of these proteins to distinct types of platinum DNA crosslinks.
- To expand the known list of proteins involved in the early cellular response to cisplatin and carboplatin DNA damage.
Main Methods:
- Synthesis of 25-base pair DNA duplexes containing either 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand crosslinks using a benzophenone-modified cisplatin analogue (Pt-BP6).
- Photocrosslinking of proteins with affinity for these platinated DNA probes in nuclear extracts from various cancer cell lines.
- Identification of photocrosslinked proteins using mass spectrometry and investigation with a longer, 90-base dumbbell probe to rule out DNA end effects.
Main Results:
- Identified DNA repair factors (RPA1, Ku70/80, Msh2, DNA ligase III, PARP-1, DNA-PKcs) and HMG-domain proteins (HMGB1-3, UBF1) binding to platinum-damaged DNA.
- Observed specific binding patterns: HMG proteins preferentially associated with the 1,2-d(GpG) adduct, while RPA1 photocrosslinked only with the 1,3-d(GpTpG) adduct.
- Demonstrated PARP-1's affinity for platinum-modified DNA for the first time and identified additional chromatin remodeling and transcription proteins using a longer DNA probe.
Conclusions:
- Provides a more comprehensive list of proteins involved in recognizing and responding to cisplatin-induced DNA damage.
- Highlights differential protein affinities for specific platinum adduct types, suggesting distinct roles in DNA repair pathways.
- Enhances understanding of the early molecular events following cisplatin and carboplatin treatment, potentially informing future cancer therapy strategies.
