ABSTRACT
Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.
Subject(s)
DNA Repair/genetics , RNA-Binding Proteins/ultrastructure , Transcription Factor TFIIH/ultrastructure , Binding Sites/radiation effects , Cryoelectron Microscopy , DNA Damage/radiation effects , DNA Helicases/genetics , DNA Helicases/ultrastructure , DNA, Single-Stranded/genetics , DNA, Single-Stranded/radiation effects , DNA, Single-Stranded/ultrastructure , DNA-Binding Proteins/genetics , DNA-Binding Proteins/ultrastructure , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , RNA-Binding Proteins/genetics , Single Molecule Imaging , Transcription Factor TFIIH/genetics , Transcription, Genetic/radiation effects , Ultraviolet Rays/adverse effects , Xeroderma Pigmentosum Group D Protein/genetics , Xeroderma Pigmentosum Group D Protein/ultrastructureABSTRACT
Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA's 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA's kinetic cycle. Measurement of UvrA's steady-state ATPase activity shows it is stimulated upon binding DNA ( kcat 0.71-1.07/s). Despite UvrA's ability to discriminate damage, we find UV-damaged DNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.-Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.
