Kinetic model for the ATP-dependent translocation of Saccharomyces cerevisiae RSC along double-stranded DNA - PubMed (original) (raw)

. 2007 Oct 30;46(43):12416-26.

doi: 10.1021/bi700930n. Epub 2007 Oct 5.

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Kinetic model for the ATP-dependent translocation of Saccharomyces cerevisiae RSC along double-stranded DNA

Christopher J Fischer et al. Biochemistry. 2007.

Abstract

The chromatin remodeling complex RSC from Saccharomyces cerevisiae is a DNA translocase that moves with directionality along double-stranded DNA in a reaction that is coupled to ATP hydrolysis. To better understand how this basic molecular motor functions, a novel method of analysis has been developed to study the kinetics of RSC translocation along double-stranded DNA. The data provided are consistent with RSC translocation occurring through a series of repeating uniform steps with an overall processivity of P = 0.949 +/- 0.003; this processivity corresponds to an average translocation distance of 20 +/- 1 base pairs (bp) before dissociation. Interestingly, a slow initiation process, following DNA binding, is required to make RSC competent for DNA translocation. These results are further discussed in the context of previously published studies of RSC and other DNA translocases.

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Figures

Figure 1

Figure 1

Time courses of ATP hydrolysis for RSC in the presence of a saturating concentration of double-stranded DNA of various lengths. The lengths of the DNA are 15, 30, 50, and 80 bp. A control experiment in which no DNA is included in the reaction is also shown.

Figure 2

Figure 2

Time course of PBP-MDCC fluorescence (_λ_ex = 430 nm, _λ_em > 450 nm) measuring Pi production during double-stranded DNA translocation by RSC along 20 and 40 bp substrates. Also shown is the time course observed during experiments conducted in the presence of 40 bp DNA and 50 _μ_g/mL heparin. Identical traces were obtained in experiments conducted in the presence of plasmid DNA and 50 _μ_g/mL heparin. All curves presented are the average of four independent traces.

Figure 3

Figure 3

Time courses of ATP hydrolysis derived from Monte Carlo computer simulations. The time courses in (A) are obtained from simulations that do not include an initial slow step in the translocation mechanism. The time courses in (B) are obtained from simulations that do include an initial slow step in the translocation mechanism. For these simulations the enzyme was assumed to translocate along the DNA at a rate of 20 bp/s with an associated dissociation rate of 1 s−1. The enzyme would hydrolyze one ATP molecule per bp translocated. The pseudo-first-order DNA binding rate constant was 100 s−1. The simulations in (B) assumed an initial slow process with an associated rate of 0.01 s−1. The lengths of the DNA used in the simulation are 20 bp (solid circles), 30 bp (solid squares), and 40 bp (open circles).

Scheme 1

Scheme 1

Scheme 2

Scheme 2

FIGURE 4

FIGURE 4

Dependence of _V_max upon double-stranded DNA length. The solid line is a NLLS fit of the data to eq 3.

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References

    1. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997;389:251–260. - PubMed
    1. Richmond TJ, Davey CA. The structure of DNA in the nucleosome core. Nature. 2003;423:145–150. - PubMed
    1. Corona DF, Tamkun JW. Multiple roles for ISWI in transcription, chromosome organization and DNA replication. Biochim. Biophys. Acta. 2004;1677:113–119. - PubMed
    1. Deuring R, Fanti L, Armstrong JA, Sarte M, Papoulas O, Prestel M, Daubresse G, Verardo M, Moseley SL, Berloco M, Tsukiyama T, Wu C, Pimpinelli S, Tamkun JW. The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol. Cell. 2000;5:355–365. - PubMed
    1. Kadonaga JT. Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines. Cell. 1998;92:307–313. - PubMed

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