Dataset Information

Structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in Nucleotide Excision Repair

ABSTRACT: TFIIH is a 10-protein complex that is conserved throughout eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). NER in eukaryotes begins by recognition of a bulky lesion by the obligate dimer Rad4-Rad23. This is followed closely by the recruitment of TFIIH which is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. This facilitates the recruitment of two exonucleases which excise the damages strand before an undamaged complement is synthesize. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3, though these later 3 are dispensable for NER.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Hee Jong Kim

LAB HEAD: Benjamin Aaron Garcia

PROVIDER: PXD021212 | Pride | 2021-07-07

REPOSITORIES: Pride

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Publications

Cryo-EM structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in nucleotide excision repair.

van Eeuwen Trevor T Shim Yoonjung Y Kim Hee Jong HJ Zhao Tingting T Basu Shrabani S Garcia Benjamin A BA Kaplan Craig D CD Min Jung-Hyun JH Murakami Kenji K

Nature communications 20210607 1

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be m ...[more]

PMID: 34099686

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Project description:The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. In addition to examining crossovers on chromosome V, we mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

Dataset Information

Structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in Nucleotide Excision Repair

Publications

Cryo-EM structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in nucleotide excision repair.

Similar Datasets

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