Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2010 Apr 2;285(14):11013-22.
doi: 10.1074/jbc.M109.094763. Epub 2010 Feb 6.

The XBP-Bax1 helicase-nuclease complex unwinds and cleaves DNA: implications for eukaryal and archaeal nucleotide excision repair

Christophe Rouillon  1 , Malcolm F White
Affiliations
Comparative Study

The XBP-Bax1 helicase-nuclease complex unwinds and cleaves DNA: implications for eukaryal and archaeal nucleotide excision repair

Christophe Rouillon et al. J Biol Chem. .

Abstract

XPB helicase is an integral part of transcription factor TFIIH, required for both transcription initiation and nucleotide excision repair (NER). Along with the XPD helicase, XPB plays a crucial but only partly understood role in defining and extending the DNA repair bubble around lesions in NER. Archaea encode clear homologues of XPB and XPD, and structural studies of these proteins have yielded key insights relevant to the eukaryal system. Here we show that archaeal XPB functions with a structure-specific nuclease, Bax1, as a helicase-nuclease machine that unwinds and cleaves model NER substrates. DNA bubbles are extended by XPB and cleaved by Bax1 at a position equivalent to that cut by the XPG nuclease in eukaryal NER. The helicase activity of archaeal XPB is dependent on the conserved Thumb domain, which may act as the helix breaker. The N-terminal damage recognition domain of XPB is shown to be crucial for XPB-Bax1 activity and may be unique to the archaea. These findings have implications for the role of XPB in both archaeal and eukaryal NER and for the evolution of the NER pathway. XPB is shown to be a very limited helicase that can act on small DNA bubbles and open a defined region of the DNA duplex. The specialized functions of the accessory domains of XPB are now more clearly delineated. This is also the first direct demonstration of a repair function for archaeal XPB and suggests strongly that the role of XPB in transcription occurred later in evolution than that in repair.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
XPB and Bax1 cooperate to cleave a model NER substrate. A, in the presence of ATP and Mg2+, the XPB-Bax1 complex cleaves a 7-nt DNA bubble substrate (Bubble 7) at three major sites located 4–6 bp 3′ of the ssDNA/dsDNA junction (black arrows). This activity is ablated when an active site residue of Bax1 is mutated (D301A; middle) and is also dependent on the activity of XPB as shown by mutation in the Walker A box of XPB (K96A; right). Control lane c, DNA alone; lane m, A + G sequence ladder. B, XPB-Bax1 cleaves the Bubble 7 substrate in the presence of ATP and magnesium, manganese, or cobalt cations. Lane C, control lane showing DNA alone. Quantification of the cleavage products yielded the following activities (relative to 100% for Mn2+): Mg2+, 24%; Ca2+, 1%; Mn2+, 100%; Co2+, 82%; Ni2+, 2%; Zn2+, 5%.
FIGURE 2.
FIGURE 2.
XPB-Bax1 cleaves a range of bubble substrates. XPB-Bax1 cleaves bubbles of 7 and 16 nt in the presence of magnesium and ATP and additionally a bubble of 3 nt in the presence of manganese and ATP. The cleavage sites are shown mapped for the three substrates. The black arrows show cleavage sites in common for all three bubbles. Cleavage further into the duplex 3′ of the bubble (white arrows) suggests further opening of the DNA by XBP.
FIGURE 3.
FIGURE 3.
Bax1 cleaves at the junction between ssDNA and dsDNA, and bubbles are extended by XPB. With the Bubble 16 substrate, Bax1 cuts at the ssDNA/dsDNA junction when XPB helicase activity is inactivated due to lack of ATP or point mutation K96A (black arrows). When XPB is active, the point of cleavage moves 4–5 nt 3′ away from the edge of the bubble, consistent with DNA strand opening by XPB (gray arrows). No activity was observed when the Bax1 nuclease was inactivated by mutation (D301A). Lane C, control, DNA alone.
FIGURE 4.
FIGURE 4.
XPB domain structure and complex formation with Bax1. A, structural model of XPB from A. fulgidus, showing the two helicase motor domains (HD1 and HD2), the N-terminal DRD, and RED motif arising from HD1 and the Thm domain arising from HD2. XPB moves in a 3′ to 5′ direction on DNA and is predicted to disrupt the DNA duplex at HD2, possibly by the physical action of the Thm domain. B, a Coomassie Blue-stained SDS-polyacrylamide gel showing purified XPB-Bax1 complex after gel filtration. The wild type and all mutant variants of XPB and Bax1 co-purify in a 1:1 complex. m, molecular weight markers; W-T, wild type XPB-Bax1; Δnuc, Bax1 D301A mutant; Δhel, XPB K96A mutant; ΔRED, mutated RED motif in XPB; ΔThm, Thm domain in XPB deleted; ΔDRD, DRD of XPB deleted.
FIGURE 5.
FIGURE 5.
Mutational analysis of XPB-Bax1 reveals subdomain function in binding and catalysis. A, activity of wild type (WT) and mutant versions of XPB-Bax1 on the Bubble 7 substrate in the presence of Mn2+ and ATP. B, activity of wild type and mutant versions of XPB-Bax1 on the Bubble 16 substrate in the presence of Mn2+, in the presence or absence of ATP. C, gel shift analysis of XPB-Bax1 binding to the Bubble 7 substrate and single-stranded DNA. The binding affinities of the wild type, ΔThm, and ΔDRD variants of XPB were compared by incubating 10 nm DNA with 50, 100, 250, and 500 nm XPB-Bax1. Lane c, DNA alone.
FIGURE 6.
FIGURE 6.
Cleavage of minimal substrates by XPB-Bax1. A, native gel electrophoresis showing cleavage of splayed duplex and overhang substrates. Oligonucleotide B50 with a 5′-fluorescein (gray hexagon) was annealed with a range of partner strands to generate minimal substrates. Approximate cleavage sites are indicated by white arrows. Cleavage products are indicated on the right of the gel. The wild type enzyme (WT) was incubated with each substrate for 5 or 20 min in the presence of ATP and MnCl2. The Δnuc mutant was incubated in the same buffer for 20 min. Control lanes are as follows. m, marker DNA showing migration of overhang and single-stranded B50 oligonucleotide; c, DNA substrate control; Δ, Δnuc mutant. B, the reaction products were also run on denaturing acrylamide TBE gels with (A + G) markers to map cleavage sites. All reactions were carried out in reaction buffer with 10 mm MnCl2 and ATP where indicated. m, A + G sequence markers; c, DNA alone; Δ, Δnuc mutant.
FIGURE 7.
FIGURE 7.
Model for XPB evolution and NER in archaea and eukarya. A, the structure of archaeal XPB revealed two helicase domains (HD1 and HD2), with an N-terminal DRD and a Thm domain arising from within HD2. There is a stable interaction with the nuclease Bax1 to form a helicase-nuclease machine. B, archaeal NER may involve duplex unwinding by XPB bound to the undamaged (bottom) strand allowing the Bax1 nuclease to cleave the damaged strand 3′ of the lesion. Further steps may be catalyzed by the archaeal XPD and XPF proteins. C, in eukarya, the interaction with Bax1 has been lost and replaced by p52, which anchors XPB to the TFIIH complex. The other TFIIH subunits required for NER are XPD, p34, p44, p62, and p8. D, a model for TFIIH binding during NER, showing XPB bound to the undamaged strand and XPD bound to the damaged strand, consistent with the archaeal scheme.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Evans E., Moggs J. G., Hwang J. R., Egly J. M., Wood R. D. (1997) EMBO J. 16, 6559–6573 - PMC - PubMed
    1. Tirode F., Busso D., Coin F., Egly J. M. (1999) Mol. Cell 3, 87–95 - PubMed
    1. Lehmann A. R. (2003) Biochimie 85, 1101–1111 - PubMed
    1. Andressoo J. O., Weeda G., de Wit J., Mitchell J. R., Beems R. B., van Steeg H., van der Horst G. T., Hoeijmakers J. H. (2009) Mol. Cell. Biol. 29, 1276–1290 - PMC - PubMed
    1. Guzder S. N., Sung P., Bailly V., Prakash L., Prakash S. (1994) Nature 369, 578–581 - PubMed

Publication types

MeSH terms