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Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
Published on: May 24, 2017
Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)
Sandra C Koch1, Jochen Kuper2, Karola L Gasteiger1
1Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians Universität München, 81377 Munich, Germany;
Abstract:
Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.
Insights
Researchers elucidated the DNA binding mechanism of the XPA protein homolog, Rad14, revealing how it recognizes DNA lesions. This finding is crucial for understanding DNA repair and xeroderma pigmentosum (XP).
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- Nucleotide excision repair (NER) removes diverse DNA lesions, and its defects cause xeroderma pigmentosum (XP).
- The function of XPA protein, critical for NER, remains unclear despite known interactions with damaged DNA.
- XPA binds kinked DNA and DNA with specific lesions, but the interaction mechanism is unknown.
Purpose of the Study:
- To elucidate the structural mechanism by which the XPA homolog, Rad14, recognizes damaged DNA.
- To understand the role of DNA structure and specific lesions in Rad14 binding.
Main Methods:
- X-ray crystallography of the Rad14 DNA-binding domain (DBD) complexed with damaged DNA.
- Analysis of DNA structures with cisplatin (1,2-GG) and acetylaminofluorene (AAF-dG) adducts.
Main Results:
- Two Rad14 molecules bind to DNA, inducing duplex melting away from the lesion.
- Each Rad14 monomer uses a β-hairpin to form a 13-mer recognition motif with a sharp DNA kink (70°) at the lesion site.
- The 1,2-GG lesion stabilizes the kink by fixing crosslinked bases at 90°, while the AAF-dG adduct intercalates to stabilize the kink.
Conclusions:
- Rad14 employs a specific binding mode involving DNA kinking and melting to recognize damaged DNA.
- Structural insights into Rad14-DNA interaction provide a mechanistic basis for XPA's role in NER.
- Understanding these interactions is key to addressing XP pathogenesis.
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