Structure and mechanism of pyrimidine–pyrimidone (6-4) photoproduct recognition by the Rad4/XPC nucleotide excision repair complex

Abstract Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine–pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4–Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an ‘open’ conformation. The MD trajectories detail how Rad4/XPC initiates ‘opening’ 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5′ partner adenine first flips out and is captured by a BHD2/3 groove, while the 3′ adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.

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Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

10.1101/2020.01.16.909549 ◽

2020 ◽

Author(s):

Debamita Paul ◽

Hong Mu ◽

Qing Dai ◽

Amirrasoul Tavakoli ◽

Chuan He ◽

...

Keyword(s):

Crystal Structure ◽

Nucleotide Excision Repair ◽

Dna Sequences ◽

Excision Repair ◽

Md Simulations ◽

Dna Duplex ◽

Open Structure ◽

Dna Structures ◽

Reverse Mode ◽

Nucleotide Excision

ABSTRACTRad4/XPC is a key DNA damage sensor for nucleotide excision repair (NER) in eukaryotes. Rad4/XPC recognizes diverse bulky lesions by flipping out two lesion-containing nucleotide pairs and inserting a β-hairpin from the BHD3 domain (β-hairpin3) into the DNA duplex. We have previously observed that Rad4 can form the same ‘open’ structure when covalently tethered to a normal DNA sequence containing consecutive C/G’s (CCC/GGG) and that a similar open-like structure can be formed even when the β-hairpin3 is lacking. Here, we report a crystal structure of the Δβ-hairpin3 mutant tethered to a sequence containing alternating C/G’s (CGC/GCG). In contrast to the previous structures, Rad4 bound to CGC/GCG in a 180°-reversed manner, capping the end of the duplex without flipping out the nucleotides. MD simulations showed that CGC/GCG was inherently less ‘openable’ than CCC/GGG and that Rad4 failed to engage with its minor groove, a hallmark of productive binding towards ‘opening’. These results reveal that DNA sequences significantly influence the thermodynamic barrier for DNA opening by Rad4, which may render certain DNA structures/sequences resistant to ‘opening’ despite a long residence time of Rad4. The reverse- mode may indicate unproductive binding for NER whereas the DNA end-binding may hint at Rad4/XPC’s functions beyond NER.

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Faculty Opinions recommendation of Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1117767.573842 ◽

2008 ◽

Author(s):

Marc Fontecave

Keyword(s):

Crystal Structure ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Nucleotide Excision

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Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4777 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4777-4788 ◽

Cited By ~ 23

Author(s):

M Baer ◽

G B Sancar

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Spectroscopic Studies ◽

Pyrimidine Dimer ◽

Nucleotide Sequence Analysis ◽

Binding Motif ◽

Pyrimidine Dimers ◽

Nucleotide Excision

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

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