Regulation of translocation polarity by helicase domain 1 in SF2B helicases

Abstract

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.

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).

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.

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.

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.

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.

Figure 6

Helicase and motor activities of the wild-type and mutant XPD helicases. (A) Histogram displaying results from helicase assays for each mutant analysed. Assays contained 2.5 μM enzyme and 50 nM forked DNA. Reactions were carried out at 45°C. Fraction unwound reflects the ratio of ssDNA product to intact forked duplex after 5 min following initiation with 4 mM ATP. Error bars represent standard deviation from the mean from a minimum of two independent experiments. (B) Rate of streptavidin displacement on 10 nM unmodified oligonucleotide by 500 nM of each mutant enzyme at RT. Biotinylated DNA was incubated for 5 min at RT with the enzyme. Translocation was initiated with 3 mM ATP and time points were taken from 0 to 10 min. Error bars represent standard deviation from the mean from a minimum of two independent experiments. (C) Rate of streptavidin displacement from modified oligonucleotides. Reactions were carried out as described for (B) but using modified oligonucleotides depicted on the left. Error bars represent standard deviation from the mean from a minimum of two independent experiments. X marks the position the modification within each oligo. Abasic oligo lacks a nucleotide at this position. The structures of the backbone modifications are cartooned. Total streptavidin displacement for each oligo and mutant is detailed in Supplementary Figure S5.

Figure 7

Model for duplex unwinding by SF2B helicases. (A) Model of TacXPD bound to a forked DNA substrate with DNA interaction positions identified. (1) Represents the initial binding or recognition point in HD2. (2) DNA passes through the central pore formed between the Arch, FeS and HD1 domains. (3) DNA binding region specific for Rad3 helicases formed by residues from HD1 and the FeS domains. (4) Proposed wedge and point of duplex separation. (B) Depiction of XPD as part of the TFIIH complex during transcription and nucleotide excision repair. (C) Extension of our binding model based upon TacXPD to other SF2B helicases, FancJ, Rtel and ChlR1. Insertions conferring substrate specificity for branched DNA substrates are outlined in red. Interaction between DNA substrates and these insertions may also be responsible for higher helicase rates and processivities typically observed in these helicases.