Translocation and stability of replicative DNA helicases upon encountering DNA-protein cross-links

Abstract

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.

FIGURE 1.

DPC-containing DNA substrates used for helicase reactions. A, cross-linked proteins, including their abbreviations and sizes. B, structures of forked DNA substrates. The substrates consisted of a 25-mer duplex containing DPC, dT₆₀, and dT₄₀ tails for helicase loading, and a ³²P label at the indicated 5′ positions. Proteins were tethered to oxanine (O) in leading and lagging template strands via an amide bond (see also Fig. 4C).

FIGURE 2.

DPCs on the translocating strand but not on the nontranslocating strand block the progression of replicative helicases. A, PAGE analysis of DnaB reaction products. Control and H2A-DPC substrates were incubated with DnaB as described in B, and products were separated by 10% native PAGE. In the substrates shown in the schemes, the black circle, asterisks, and oval rings indicate DPC, a ³²P label, and helicase, respectively. B, time courses of DNA unwinding by DnaB (a), T7gp4 (b), Mcm467 (c), and Tag (d) with substrates containing DPCs in the leading template strand (leading-DPC, left graphs) and the lagging template strand (lagging-DPC, right graphs). In a–d, DNA substrates (2 fmol) were incubated with DnaB (50 ng), T7gp4 (50 ng), Mcm467 (50 ng as total protein), or Tag (15 ng) at 37 °C for the indicated times. The percentages of unwound products relative to the total substrates are plotted against the reaction time. The data are the average of two independent experiments. The abbreviations next to the plotted lines indicate cross-linked proteins (see Fig. 1A). The reactions with control templates without DPCs (Cont) are shown by dotted lines and open circles.

FIGURE 3.

The critical size of DPCs for clearance by replicative helicases is 5.0–14.1 kDa. The percentages of unwound products relative to the total substrates after a reaction time of 15 min (Fig. 2B) are plotted against the sizes of cross-linked proteins (Fig. 1A). The results with leading- and lagging-DPC substrates are represented by open and closed circles, respectively. The numbers on the graphs indicate the critical sizes of cross-linked proteins (in kDa) for clearance by respective helicases.

FIGURE 4.

Orientation of cross-linked proteins may affect the translocation of helicases through DPCs. A, effect of prolonged helicase reactions on the yield of unwound products. The helicase reactions were performed as described in Fig. 2B except that the reaction time was extended to 40 min. The percentages of unwound products relative to the total substrates are plotted against the reaction time. The abbreviations next to plotted lines indicate cross-linked proteins (see Fig. 1A). B, effect of the increasing amounts of helicases on the yield of unwound products. The helicase reactions were performed as described in Fig. 2B except that increasing amounts of helicases were used. MID- and H2A-DPCs on the lagging template strand were used for DnaB and T7gp4, whereas those on the leading template strand were used for Mcm467 and Tag. The percentages of unwound products are plotted against the amount of helicases. The data in A and B are based on a single experiment. C, reaction scheme for oxanine and a protein that results in two orientations of cross-linked protein. The amino groups of lysine residues involved in cross-linking are shown by “–NH₂.” D, schemes showing that cross-linked proteins in nonblocking and blocking orientations differentially affect the translocation of replicative helicases through DPC.

FIGURE 5.

Replicative helicases stalled by DPC dissociate from DNA with a half-life of 15–36 min. A, PAGE analysis of DNA-stalled helicase complexes. DNA substrates containing NEI-DPC on the translocating strand (2 fmol) were incubated with DnaB (50 ng) or Mcm467 (100 ng as total protein). For analysis of the dissociation kinetics of stalled helicases, a competitor (100 fmol of cold control DNA substrate without DPC) was added to the reaction mixture at 20 min to quench the loading of helicases onto the DPC substrate. The complexes were stabilized by Sulfo-EGS every 10 or 20 min and analyzed by 10% native PAGE (lower gels for DnaB and Mcm467). Upper gels for DnaB and Mcm467 show PAGE data obtained in the reactions without a competitor. B, changes in the amount of DNA-stalled helicase complexes with incubation time. The percentages of DNA-stalled helicase complexes relative to the total DPC substrates are plotted against the incubation time: filled circles, without a competitor; open circles, with a competitor. The data are means ± S.D. (error bars) based on three or four independent experiments. C, stabilities of DNA-stalled helicase complexes. The half-lives were evaluated from the decay of complexes after addition of the competitor (open circles in B) by assuming exponential decay.

FIGURE 6.

Possible fates of replisomes that encounter conventional bulky damage and DPCs. A, fates of the replisome that encounters conventional bulky damage on the translocating (left) and nontranslocating (right) strands of the helicase. B, fates of the replisome that encounters a DPC on the translocating (left) and nontranslocating (right) strands of the helicase. Note that the scheme was drawn for eukaryotic replication, where the replicative helicase translocates on the leading template strand.