Regulation of translocation polarity by helicase domain 1 in SF2B helicases
2011; Springer Nature; Volume: 31; Issue: 2 Linguagem: Inglês
10.1038/emboj.2011.412
ISSN1460-2075
AutoresRobert A. Pugh, Colin G. Wu, Maria Spies,
Tópico(s)RNA modifications and cancer
ResumoArticle11 November 2011Open Access Regulation of translocation polarity by helicase domain 1 in SF2B helicases Robert A Pugh Robert A Pugh Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Colin G Wu Colin G Wu Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Maria Spies Corresponding Author Maria Spies Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Robert A Pugh Robert A Pugh Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Colin G Wu Colin G Wu Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Maria Spies Corresponding Author Maria Spies Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Author Information Robert A Pugh1,2, Colin G Wu1 and Maria Spies 1,2,3 1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA 2Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA *Corresponding author. Department of Biochemistry, University of Illinois at Urbana-Champaign, RAL493, 600 S. Mathews Avenue, Urbana, IL 61801-3602, USA. Tel.: +1 217 244 9493; Fax: +1 217 244 5858; E-mail; [email protected] The EMBO Journal (2012)31:503-514https://doi.org/10.1038/emboj.2011.412 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Structurally similar superfamily I (SF1) and II (SF2) helicases translocate on single-stranded DNA (ssDNA) with defined polarity either in the 5′–3′ or in the 3′–5′ direction. Both 5′–3′ and 3′–5′ translocating helicases contain the same motor core comprising two RecA-like folds. SF1 helicases of opposite polarity bind ssDNA with the same orientation, and translocate in opposite directions by employing a reverse sequence of the conformational changes within the motor domains. Here, using proteolytic DNA and mutational analysis, we have determined that SF2B helicases bind ssDNA with the same orientation as their 3′–5′ counterparts. Further, 5′–3′ translocation polarity requires conserved residues in HD1 and the FeS cluster containing domain. Finally, we propose the FeS cluster-containing domain also provides a wedge-like feature that is the point of duplex separation during unwinding. Introduction Helicases are motor proteins that use the chemical energy of NTP binding and hydrolysis to move directionally on nucleic acid lattices. Translocation polarity is a predefined, inherent feature for each particular enzyme and reflects helicase movement either in the 3′–5′ or in the 5′–3′ direction (Singleton et al, 2007). Cellular activities of all bona fide DNA helicases stem from their directional translocation. Defining the mechanism by which superfamily II (SF2) helicases adopt translocation polarity has proven elusive due to the absence of structural information on the complex of a 5′–3′ helicase (SF2B) bound to DNA. Superfamily I (SF1) and SF2 helicases translocate in either 3′–5′ (denoted as SF1A and SF2A) or 5′–3′ (SF1B and SF2B) direction using structurally identical motor cores defined by similar sets of conserved helicase signature motifs (Singleton et al, 2007; Fairman-Williams et al, 2010). Recent structural studies suggested the mechanism underlying polarity of translocation in SF1 helicases (Singleton et al, 2004; Saikrishnan et al, 2008, 2009). The motor core of SF1A enzymes consists of two RecA-like folds, 1A and 2A (Figure 1A), where domain 1A contacts the 3′-end of the occluded ssDNA and domain 2A faces the 5′-end (Figure 1A, top). It has been accepted that all SF1A helicases bind DNA with the same orientation and translocate using the same sequence of conformational changes within the motor regulated by binding and hydrolysis of ATP (Soultanas and Wigley, 2000). The translocation strand bound in the cleft, which spans both domains 1A and 2A, makes extensive contacts with residues interacting with the bases of the bound nucleotide. Figure 1.Orientation of SF1 and SF2 helicases bound to ssDNA: (A) Schematic representation of the mechanism underlying polarity of SF1 helicases. Upon binding to ssDNA, domain 2A of SF1 helicases interacts with the 5′-end of occluded ssDNA and 1A interacts with the 3′-end. These enzymes translocate in either the 3′–5′ direction with HD2 as the leading fold or 5′–3′ with 1A being the leading fold. (B) SF2A helicases translocate 3′–5′ with the same orientation as SF1 helicases. In order to translocate in the 5′–3′ direction, the helicase will either bind DNA with the same orientation and reverse the conformational change between the motor domains (middle), or it may bind to DNA with the opposite orientation but maintain the relative orientation of the motor subunits with HD2 being the leading fold (bottom). (C) Primary structure (top) and domain organization (bottom) of XPD helicase from T. acidophilum. Modular domains are colour coded as HD1 (blue), HD2 (green), Arch (purple) and FeS (orange). Modular insertions characteristic to eukaryotic ChlR1, FancJ and Rtel helicases are indicated by red rectangles. Helicase signature motifs are shown as black bars and roman numerals on the primary structure. Motifs directly involved in DNA binding are highlighted in black on the ribbon representation of TacXPD (pdb: 2VSF). Download figure Download PowerPoint The binary complex of RecD from D. radiodurans (SF1B helicase) bound to an 8mer ssDNA revealed that the helicase interacts with the first four nucleotides of the 5′-end using domain 2A while the four nucleotides of the 3′-end interact with the 1A domain as depicted in Figure 1A (bottom) (Saikrishnan et al, 2009). As a result, both SF1A and SF1B helicases bind to the translocating strand with the same orientation. Polarity is the result of a predefined sequence of the conformational changes induced by ATP binding and hydrolysis, which is reversed in the SF1B enzymes compared with SF1A. One significant difference between SF1A and SF1B helicases bound to ssDNA is that the key residues involved in translocation interact with the backbone of DNA for SF1B as opposed to base interactions observed for SF1A. For SF1 enzymes, a distinction can be made between 3′–5′ and 5′–3′ enzymes in signature helicase motif Ia, which contains a conserved phenylalanine for SF1A helicases. A proline found in the corresponding position in SF1B enzymes opens a binding pocket to be occupied by a base from the translocating strand (Saikrishnan et al, 2008). A proline in this motif is also found in SF2A helicases, however, a conserved arginine and threonine are found in motif Ia in SF2B helicases (Pugh et al, 2008a). Additionally, the two highly conserved threonine residues typically found in motifs Ic and V of SF1 and SF2 helicases that separate nucleic acid duplexes by translocation are absent in SF2B helicases (Fairman-Williams et al, 2010). Similarly to SF1 enzymes, the motor core of SF2 helicases comprise two RecA-like folds, helicase domain 1 (HD1) and helicase domain 2 (HD2) which are equivalent to helicase domains 1A and 2A, respectively (Figure 1B; Singleton et al, 2007; Fairman-Williams et al, 2010). Several structures are currently available for SF2A helicases bound to nucleic acids (Kim et al, 1998; Buttner et al, 2007; Luo et al, 2008) and for dsDNA translocating motors (Thoma et al, 2005). These structures show HD2 bound to the 5′-end of the oligonucleotide and HD1 to the 3′-end (Figure 1B, top). Only apo structures are currently available for SF2B enzymes (Fan et al, 2008; Liu et al, 2008; Wolski et al, 2008). Identifying the orientation of XPD bound to DNA is a prerequisite for establishing how directional translocation is determined in SF2 helicases. Two mutually exclusive models can be proposed to explain reversal of translocation polarity in SF2B helicases (Figure 1B). A helicase may bind ssDNA with the same orientation as SF1 and SF2A helicases, then translocate in the opposite direction by reversing the ATP-driven conformational changes within the motor (Figure 1B, middle). Alternatively, a 5′–3′ helicase would bind to DNA with the opposite orientation compared with SF1A and SF2A helicases and translocate in the opposite direction by maintaining the same molecular motor movement as SF2A enzymes (Figure 1B, bottom). The first model is consistent with the mechanism by which translocation polarity is reversed in SF1 helicases. The second model is consistent with the difference between motif 1a in SF2B helicases compared with that of the SF1A enzymes. In contrast to their SF1 counterparts, both SF2A and SF2B helicases are believed to interact with the phosphodiester backbone of their respective lattices. This has been demonstrated for SF2A helicases through structural studies (Kim et al, 1998; Buttner et al, 2007; Luo et al, 2008) and inferred for SF2B enzymes from their ability to bypass damaged bases (Rudolf et al, 2010) and ssDNA binding proteins that specifically interact with the bases (Honda et al, 2009). Only a limited number of SF2B helicases have been identified, all of which belong to the Rad3 family comprising XPD (Rad3), Rtel, FancJ, DinG and ChlR1 (White, 2009; Wu et al, 2009). All helicases in this family are involved in DNA repair and in maintenance of the genetic integrity. Here, we used XPD from Thermoplasma acidophilum to determine how polarity is defined in SF2 helicases. In humans, XPD is one of the two helicases of the TFIIH complex, which is involved in transcription initiation and nucleotide excision repair (NER; Egly and Coin, 2011). The helicase activity of XPD is dispensable for its role in transcription initiation (Dubaele et al, 2003). It, however, is required for NER (Coin et al, 2007). XPD is composed of four domains (Fan et al, 2008; Liu et al, 2008; Wolski et al, 2008). The modular motor core consists of HD1 and HD2. Two additional domains, FeS and Arch, are found within HD1 (schematically depicted in Figure 1C). The FeS domain is stabilized by the presence of a 4Fe-4S cluster and the Arch domain represents a unique structural fold. The Arch domain sits above the two helicase motor domains, and forms a donut-like structure with a central pore between the FeS domain and HD1 where ssDNA is proposed to pass (Fan et al, 2008; Liu et al, 2008; Wolski et al, 2008). Previously, we suggested that XPD binds to a forked DNA or to its natural bubble-like substrate so that the FeS domain is involved in strand separation (Pugh et al, 2008a). Implicit in this model was XPD binding the translocating strand with the same orientation as SF1 and SF2A helicases. Two mutually exclusive orientations of ssDNA, however, were proposed based on the crystal structures (Fan et al, 2008; Liu et al, 2008; Wolski et al, 2008). Here, we set out to provide direct evidence for bound ssDNA orientation and to identify the path of the translocation strand through the helicase. To test these two proposed translocation models, we probed XPD–ssDNA complex using Fe(III) (s)-1-(p-bromoacetamidobenzyl) ethylenediamine tetraacetic acid (FeBABE), a hydroxyl radical generating moiety that was site specifically incorporated into 12mer ssDNA. Using the reverse footprinting technique (reviewed in Meares et al, 2003), we have determined that polarity in SF2 helicases is accomplished similarly to SF1 helicases. After confirming the binding orientation, we hypothesized that contacts from HD1 and the FeS domain with the translocating strand of the DNA substrate would play an important role in controlling XPD translocation. Mutational analysis was conducted to identify key residues in domains HD1 and FeS of XPD and their influence on duplex unwinding activity was examined. We have identified two aromatic residues near the FeS cluster that may serve as a wedge for initiating duplex separation. Finally, five positively charged residues and a histidine contributed by FeS and HD1 domains positioned to interact with the 3′-end of the translocating strand are important for either promoting or inhibiting substrate unwinding by XPD helicase, implicating these residues in the interaction with the translocating strand on its path between the motor core and duplex separation site. The model is set forward whereby these interactions restrict XPD processivity distinguishing it from more processive helicases in the family (such as FancJ and Rtel). Results FeBABE mediated proteolytic cleavage of XPD helicase In order to determine the orientation of DNA when bound by XPD, the artificial protease, FeBABE, was chemically incorporated into a DNA oligonucleotide containing phosphorothioate linkages positioned along the DNA backbone during oligonucleotide synthesis creating a proteolytic DNA (Schmidt and Meares, 2002). In the presence of ascorbic acid and H2O2, a hydroxyl radical is generated. The hydroxyl radical diffuses and cleaves the peptide backbone generating protein cleavage fragments. Cleavage sites can be assigned with high precision by comparing mobility of the FeBABE-produced fragments with chemical digests generated from the same protein (Owens et al, 1998). TacXPD helicase containing an N-terminal 6 × His tag and a C-terminal FLAG tag was purified to homogeneity (Supplementary Figure S1A). A series of 12mer ssDNA oligonucleotides were labelled with FeBABE at a single position on the backbone in between bases as numbered from the 5′-end. Protein fragments were resolved by SDS–PAGE and visualized by western blots to map the distance between the two termini of the XPD polypeptide and the cleavage sites (Figure 2A and B; Supplementary Figure S2A and B). Each fragment was assigned a residue based upon its molecular weight as determined by the distance migrated on the gel as described in Materials and methods (Supplementary Table S1). Figure 2.FeBABE generated fragments. Bands were detected by western blot using α-6 × his (N-terminus) antibodies in (A) and α-FLAG (C-terminus) antibodies in (B). (A) α-His (N-terminus) cleavage pattern resulting from FeBABE mediated hydroxyl radical cleavage of XPD. Oligonucleotides containing FeBABE are represented by arrows (5′–3′) positioned above the figures. Positions of FeBABE are indicated by •) above the gels. Chemical digests of XPD cleaved at methionines and cysteines were used for precise identification of the size for each fragment (•) as determined from migration distance on the gel. The linear relationship of the log10 of the molecular weight of known fragments from the chemical digests was fitted with a straight line and cleavage fragments are indicated. (B) α-Flag (C-terminus) cleavage pattern resulting from FeBABE mediated hydroxyl radical cleavage and the chemical digests used to determine the molecular weights of each indicated fragment. Download figure Download PowerPoint FeBABE mediated cleavage indicates the orientation of XPD bound to DNA Using a series of 12mer ssDNA substrates each containing a single FeBABE modification (schematically depicted over corresponding lanes in Figure 2 and Supplementary Figure S2), we expected to observe a change in the unique fragments generated by hydroxyl radicals. The length of the FeBABE-conjugated DNA substrates was shorter than 20 nucleotides occluded by primary and secondary DNA binding sites of XPD helicase (Honda et al, 2009) to ensure that we achieve the cleanest distribution of cleavage sites. When mapped on the structure of XPD, the change in the cleavage pattern should reveal the orientation of XPD when bound the substrate, and the path of DNA through the helicase. Cuts generated using substrates with FeBABE near the 5′-end of ssDNA appear in HD2 and in the Arch domain above HD2 (Figure 3A), placing the 5′-end of DNA in contact with HD2. Specifically, substrate (1) produced fragments on α-helix 22 in HD2 and β-strand 7 in the Arch domain; α-helix 22 in HD2 forms the floor of the channel where the translocation strand is expected to bind as the helicase translocates away from the 5′-end; β-strand 7 is located above α-helix 23 forming the top of the channel for ssDNA binding; substrates (2) and (3) produced cuts in the N-terminal region of α-helix 24, while substrates (1) and (4) cut on the C-terminal end of α-helix 24 on helicase motif VI (Figure 3A); α-helix 24 runs parallel to helicase motifs V, which is known to interact with DNA (Pyle, 2008) and motif Va which has been implicated in DNA binding (Liu et al, 2008). The position of these cuts is consistent with the 5′-end of the translocating strand interacting with highly conserved residues found within the DNA binding motifs as they map directly adjacent to these motifs. No cuts were observed in HD1 or FeS domain. Figure 3.FeBABE generated fragments mapped to XPD. Positions of FeBABE within each set of oligonucleotides are indicated by colour coded asterisks and numbers. Fragments are colour coded with the position of FeBABE from which they were generated as they are mapped on the structure of TacXPD helicase (pdb: 2VSF). Helicase motifs involved in binding to DNA are indicated in black. HD1 appears in light blue. HD2 is coloured light green. The Arch domain appears in dark grey and the FeS domain appears in light grey. (A) Fragments generated from FeBABE incorporated at the 5′-end of the substrate. (B) Fragments mapped from centrally located FeBABE substrates. (C) Fragments mapped on XPD from 3′-end labelled substrates. Download figure Download PowerPoint Hydroxyl radical cleavage resulting from centrally positioned FeBABE yielded cuts in all four domains (Figure 3B). A majority of the cuts were observed in HD2. Cleavage products were mapped on α-helix 19 for substrates (5–7) and α-helix 20 for substrates (5) and (6). These two helices sandwich helicase motif IV, a DNA binding motif (Pyle, 2008). Substrates (5–7) also cleave the protein on the loop between α-helices 20 and 21 adjacent to helicase motif IV. α-Helix 21 runs along the ATP binding cleft between HD2 and HD1. N-terminally, this helix is cleaved by substrates (5) and (7) while C-terminal cuts are generated by (5) and (6). The loop between α-helix 21 and β-strand 14 is also cleaved by substrates (5) and (6). A loop running between β-strand 15 and α-helix 22 that contains helicase motif Va implicated in DNA binding is cleaved by substrates (5) and (6). Substrate (7) produces additional cuts in α-helix 22 described above. Substrates (5) and (6) also cleave the N-terminal region of α-helix 24 where cuts from substrates (2) and (3) are also observed. Complexes of other SF1 and SF2 helicases with DNA suggest that the translocating strand spans the cleft between HD1 and HD2. Fragments from substrates (5–7) mapping to the Arch domain indicate that this is also true for SF2B helicases (Figure 3B). The Arch domain is centrally located above the two RecA-like folds. Cleavage sites identified within this domain were mapped on the ceiling of the anticipated channel above the interface between the motor domains. All three substrates cleave the loop leading into β-strand 7, as well as, β-strand 9 leading into α-helix 17, which sits above the central channel. Finally, the three substrates also cleave α-helix 12 above the central channel for DNA adjacent to HD1. The FeS domain is an insertion within HD1 stabilized by the presence of an 4Fe-4S cluster (Rudolf et al, 2006; Fan et al, 2008; Liu et al, 2008; Wolski et al, 2008). Two cuts, one generated by substrate (6) and the other by substrate (7) both map to α-helix 5 in the FeS domain (Figure 3B). This helix is located on the entrance side of the central pore formed between the Arch domain, FeS domain and HD1. One of the conserved cysteines (Cys113) essential for the FeS cluster integrity and therefore for translocation is located within this helix. FeBABE positioned at the 3′-end of DNA (substrates 8–11) yields fewer cuts in HD2 and more cuts in the FeS domain and HD1 (Figure 3C). Cuts in HD2 map to α-helix 20 and the loop leading into α-helix 20. Cuts mapped to the Arch domain appear in the loop above HD2 between α-helix 13 and β-strand 7. α-Helix 12 is cleaved by all four substrates containing FeBABE close to the 3′-end. This helix sits above the central ssDNA binding channel and forms a part of the central pore through which the translocating strand exits the helicase motor domains as the helicase translocates in the 5′ direction. On the other side of the central pore, the N-terminal loop leading into α-helix16 and α-helix16 itself is cleaved by substrates (9–11). Substrates (9–11) also generated two cleavage sites mapped to HD1 (Figure 3C). These cuts were in β-sheet 5 leading into α-helix 11 containing the DEAH residues of the conserved helicase motif II. All four of the 3′-end labelled substrates cleaved α-helix 5 as the 3′-end passes through the central pore as described above for substrates (5–7). In summary, FeBABE footprinting indicates that XPD binds to ssDNA with the same orientation as SF1 and SF2A helicases. HD2 interacts with the 5′-end of DNA while HD1 interacts with the 3′-end. The large number of fragments generated in HD2 when FeBABE was moved towards the 3′-end of DNA is consistent with the mechanism whereby XPD binding to ssDNA is initiated at the 5′-end of occluded region through recognition by residues in HD2. This allows ssDNA to bind in a number of different registers, which can result in a free and floppy 3′-end capable of generating fragments in all domains of the helicase. Fluorescence footprinting confirms orientation of XPD on ssDNA To confirm the results of the FeBABE cleavage experiments, we took advantage of our previously reported observation that the FeS cluster of XPD helicase quenches fluorescent dyes and that the magnitude of fluorescence quenching decreases with distance between the dye and the FeS cluster (Honda et al, 2009). We expected that the polarity of bound ssDNA will be reflected in the quenching trend of the Cy5 dye site specifically incorporated into ssDNA substrates. Binding titrations revealed that the total magnitude of quenching measured over the course of the reactions increased as the Cy5 fluorophore was placed closer to the 3′-end compared with the substrates containing fluorophore closer to the 5′-end (Figure 4). Binding curves (Figure 4A) obtained when Cy5 was positioned at either the 5′ or 3′ terminus closely resemble single site binding curves, whereas binding of XPD to centrally positioned Cy5-labelled oligonucleotides appeared more complex. It is possible that at high protein concentrations more than one XPD molecule was bound to these substrates resulting in non-saturable quenching. Therefore, the comparisons were made at the XPD concentration (50 nM) corresponding to the inflection point in the binding isotherms. Comparing the data for each substrate obtained in the presence of 50 nM XPD showed that the greatest magnitude of quenching (≈65%) was observed when the Cy5 fluorophore was positioned between nucleotides 11 and 12 at the 3′-end (Figure 4B). Quenching was at its lowest point (≈12%) when Cy5 was incorporated between nucleotides 1 and 2 at the 5′-end (Figure 4B). This confirms the orientation of the helicase revealed by the FeBABE data showing that the 5′-end of DNA is located furthest away from the FeS cluster. Figure 4.Orientation of XPD binding determined by Cy5 quenching. (A) Cy5 was site specifically incorporated into 12mer ssDNA as indicated by open circles on each oligonucleotide. Binding of XPD to six 12mers containing Cy5 at different locations was monitored by following XPD-dependent quenching of Cy5 fluorescence. (B) Histogram of the total quenching observed at 50 nM XPD and 10 nM Cy5-labelled ssDNA. Titrations were carried out in duplicate. Download figure Download PowerPoint HD1 and the FeS cluster-containing domain contact the translocating strand during duplex unwinding Given the orientation of XPD when bound to the translocating strand, HD1 and the FeS cluster-containing domain must be at the point of duplex separation, and therefore are expected to contact both the translocating strand and the displaced strand. We hypothesized that a number of positively charged residues and a histidine located on the opposite side of the donut-like hole formed between the HD1, Arch and FeS domains would be responsible for contacting either the displaced strand or the translocating strand (Figure 5). Each of these residues was individually mutated to alanine and subsequently purified to homogeneity (Supplementary Figure S1B). After purification, an FeS cluster was found in all mutants except for C113A which provides one of the required cysteine ligands for the cluster (Supplementary Figure S3A). Purified R88A, K117A and Y130A/F131A mutants have a different shade of colour indicating a compromised FeS cluster (Supplementary Figure S3A). Proper folding of each mutant was confirmed by CD spectroscopy (Supplementary Figure S3B). All purified enzymes were ssDNA-dependent ATPases (Table I). Efficiency of ATP hydrolysis (kcat/KM) was reduced >20-fold in R88A and C113A mutant enzymes and nearly eight-fold in Y130A/F131A compared with WT (Table I; Supplementary Figure S4A). Binding affinities for both ssDNA and forked DNA were determined using Cy5 quenching as a reporter of bound state for all enzymes except for C113A (which does not contain FeS cluster and therefore does not quench fluorescence) (Table I; Supplementary Figure S4B and C). The binding titrations for K117A and Y130A/F131A resulted in unique curves that may have resulted from the different colour of the enzyme. As a consequence, binding affinity was determined by fitting the data between 0 and 50 nM or 100 nM XPD as indicated in Table I. Figure 5.Residues located in the FeS domain and HD1 affect helicase and streptavidin displacement activities of XPD. Surface rendition of FeS, Arch and HD1 domains of XPD. Residues implicated in binding to the translocating strand and involved in duplex separation (black) were replaced with alanine. Download figure Download PowerPoint Table 1. ATPase and binding affinity for XPD and mutants ATPase Binding kcat, s−1 KM, μM kcat/KM, s−1M−1 ssDNA Kd, nM Fork Kd, nM WT 315±15 235±42 1.34 × 106 13.7±1.6 8.5±2.9 88 65±11 970±392 6.67 × 104 10.2±1.7 9.1±2.6 113 87±27 1357±909 6.42 × 104 ND ND 116 263±13 184±36 1.43 × 106 13.7±1.6 18.9±3.7 117 200±32 194±122 1.04 × 106 2.8±0.9a 11.4±4.7a 118 278±13 679±79 4.10 × 106 14.9±1.9 14.9±2.8 130 198±54 820±583 2.42 × 105 11.0±1.4 11.5±3.7 131 183±29 654±285 2.79 × 105 10.6±1.5 17.7±4.1 130/131 140±38 792±568 1.77 × 105 4.7±0.8a 9.1±5.2b 170 328±42 402±170 8.17 × 105 42.9±3.7 22.6±2.8 194 334±6 290±18 1.15 × 106 31.7±2.8 30.8±3.3 198 322±14 185±31 1.74 × 106 11.5±2.5 4.5±1.7 170/194 301±7 341±26 8.82 × 105 18.3±3.0 21.0±3.4 170/198 336±16 615±77 5.46 × 105 9.5±1.6 29.6±4.1 194/198 276±14 232±39 1.19 × 106 9.6±1.2 21.2±3.7 170/194/198 252±20 596±123 4.23 × 105 19.8±2.4 26.3±3.0 Determined using only the data collected between 0 and 50 nM of XPD. Determined using only the data collected between 0 and 100 nM of XPD. WT enzyme bound ssDNA with KD=13.7 nM. Several mutant enzymes had altered binding affinity for ssDNA. Affinity of K170A was nearly four-fold lower than that of the wild type, and three-fold lower affinity was observed for K194A. When these two residues were mutated together binding affinity was similar to WT. The triple mutant, K170A/K194A/H198A bound ssDNA nearly 1.5-fold less tightly than the WT. Binding affinities were determined for each mutant in the absence of sodium chloride for a forked DNA substrate (Table I; Supplementary Figure S4C). WT, R88A, K117A, Y130A, Y130A/F131A and H198A had similar binding affinity (Table I). Approximately, two-fold or more reduction in binding affinity was observed for R116A, F131A, K170A, K194A, K170A/K194A, K170A/H198A, K194A/H198A and K170A/K194A/H198A. Collectively, these data confirmed that predicted residues do indeed participate in the interaction with DNA. Secondary DNA binding site controls force generation, DNA translocation and unwinding by XPD Translocation, ability to generate force and unwinding activity of mutant XPD enzymes were used to analyse the role of individual residues found in HD1 and the FeS domain of XPD (Figure 6). Translocation and force generation by the wild-type and mutant XPD enzymes were inferred from their ability to facilitate streptavidin displacement from ssDNA oligonucleotide biot
Referência(s)