Single-stranded DNA binding activity of XPBI, but not XPBII, from Sulfolobus tokodaii causes double-stranded DNA melting.

State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Rd., Jinan 250100, People's Republic of China.
Extremophiles (Impact Factor: 2.2). 01/2011; 15(1):67-76. DOI: 10.1007/s00792-010-0338-z
Source: PubMed

ABSTRACT XPB helicase is the largest subunit of transcription factor IIH (TFIIH), a ten-subunit protein complex essential for transcription initiation and nucleotide excision repair (NER) in Eukarya. Two XPB homologues (XPBI and XPBII) are present in the genome of most crenarchaeota, one of the two major phyla of archaea; however, the biochemical properties have not been fully characterized and their cellular roles have not been clearly defined. Here, we report that XPBI from the hyperthermophilic crenarchaeon Sulfolobus tokodaii (StoXPBI) is able to destabilize double-stranded DNA (dsDNA) helix independent of ATP (designated as dsDNA melting activity). This activity is inhibited by single-stranded DNA (ssDNA) and relies on the unique N-terminal domain of StoXPBI, which is also likely responsible for the intrinsic strong ssDNA binding activity of StoXPBI as revealed by deletion analysis. We demonstrate that the ATPase activity of StoXPBII is remarkably stimulated by StoBax1, a nuclease partner of StoXPBII. The role of the unique dsDNA melting activity of XPBI in NER in archaea was discussed.

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    ABSTRACT: Helicases must unwind DNA at the right place and time to maintain genomic integrity or gene expression. Biologically critical XPB and XPD helicases are key members of the human TFIIH complex; they anchor CAK kinase (cyclinH, MAT1, CDK7) to TFIIH and open DNA for transcription and for repair of duplex distorting damage by nucleotide excision repair (NER). NER is initiated by arrested RNA polymerase or damage recognition by XPC-RAD23B with or without DDB1/DDB2. XP helicases, named for their role in the extreme sun-mediated skin cancer predisposition xeroderma pigmentosum (XP), are then recruited to asymmetrically unwind dsDNA flanking the damage. XPB and XPD genetic defects can also cause premature aging with profound neurological defects without increased cancers: Cockayne syndrome (CS) and trichothiodystrophy (TTD). XP helicase patient phenotypes cannot be predicted from the mutation position along the linear gene sequence and adjacent mutations can cause different diseases. Here we consider the structural biology of DNA damage recognition by XPC-RAD23B, DDB1/DDB2, RNAPII, and ATL, and of helix unwinding by the XPB and XPD helicases plus the bacterial repair helicases UvrB and UvrD in complex with DNA. We then propose unified models for TFIIH assembly and roles in NER. Collective crystal structures with NMR and electron microscopy results reveal functional motifs, domains, and architectural elements that contribute to biological activities: damaged DNA binding, translocation, unwinding, and ATP driven changes plus TFIIH assembly and signaling. Coupled with mapping of patient mutations, these combined structural analyses provide a framework for integrating and unifying the rich biochemical and cellular information that has accumulated over forty years of study. This integration resolves puzzles regarding XP helicase functions and suggests that XP helicase positions and activities within TFIIH detect and verify damage, select the damaged strand for incision, and coordinate repair with transcription and cell cycle through CAK signaling.
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