Biochemistry 1987, 26, 2001-2009
200 1
Measurement of Interstrand Cross-Link Frequency and Distance between Interruptions in DNA Exposed to 4,5',8-Trimethylpsoralen and Near -Ultr aviolet Light? Noboru Matsuof and Peter M. Ross* The Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York 10021 -6399 Received July 14, 1986; Revised Manuscript Received October 31, 1986
ABSTRACT: Bifunctional psoralens react photochemically with D N A to form single-strand adducts and interstrand, chemical cross-links. Cross-link formation is first order with [PI, the concentration of added psoralen, when [PI 5 x 10-5 460 666 950 "Assuming [SI = 0.3[DNA]; see text. b K d = 1.3 X IO-' mol/L; [8-MOP] = 10" mol/L. cKd = 5.6 X mol/L; [TMP] = mol/L. Concentration of ligand above which more than 5% of bindmol/L. ing sites are saturated. e [TMP] = 1.7 X
molecular weight distribution. We then tested our model and compared the results with those from published procedures. THEORY Binding of Psoralen to DNA. Psoralens intercalate between base pairs of duplex nucleic acid (Dall'Acqua et al., 1978, 1979a; Isaacs et al., 1977). The dissociation constant for this "dark", noncovalent binding of psoralen to DNA is defined as Kd = [pfl Is,] / where [P,] is the concentration of free psoralen, [S,] is the concentration of unoccupied binding sites, and [PSI is the concentration of bound psoralen. If [SI and [PI are the total concentration of binding sites and added psoralen, then [SI = [S,] [PSI and [PI = [P,] + [PSI. Solving for [PSI
+
[PSI = %{[PI + [SI + Kd d [ P l 2 - 2([s1 - Kd)[pl + ([SI + Kd)') (1) Interstrand cross-links form in proportion to [PSI (Dall'Acqua
0006-2960/87/0426-2001~01.50/0 0 1987 American Chemical Society
2002
MATSUO AND ROSS
BIOCHEMISTRY
0.6
weight distribution is neither uniform nor random (Rubenstein et al., 1961; Harrington & Zimm, 1965). Sheared DNA of average length E. has i length groups. If pi is the fraction of total DNA in the jth group, lj is the length in base pairs of jth group molecules, and Fj is the fraction of DNA free from cross-links in lj base pair long DNA
0.4
(3)
1.0
0.8
Lr
The slope in a plot of F against log [PI for DNA mixtures in eq 3 will be shallower than that for DNA of a single molecular weight because of the various length components. If we define
0.2
0 0
50
100
150
micrograms DNA
i
y = -In F = -In Cpje-k',[Pl j= 1
ml FIGURE 1: Effect of [DNA] on psoralen photo-cross-linkage: solid lines and X's, TMP dotted lines and circles, 8-MOP. Phage X DNA diluted serially in TE buffer to the concentrations shown on the abscissa was incubated with 1.8 X lo-* mol/L TMPor with 1.1 X 1W5mol/L 8-MOP and exposed to 4 W/m2 360-nm light. Samples were diluted in TE buffer to 14.4 pg/mL (or left at 7.2 pg/mL), denatured in alkali, and tested for S1 sensitivity as described under Materials and Methods. Un-cross-linked DNA of the same concentration was denatured and digested with S1 as a control.
et al., 1979b). When [PSI is proportional to [PI, photobinding can be predicted from [PI, which is much easier to measure than [PSI. We tested the linearity of eq 1, using 1.3 X mmol/L for 8-methoxypsoralen (8-MOP) (Rodighiero & Dall'Acqua, 1984) and 5.6 X mol/L for 4,5',8-trimethylpsoralen (TMP) (Isaacs et al., 1977) as dissociation constants. In Table I, [PS]/[S] was calculated from eq 1. Columns 3 (for 8-MOP) and 4 (for TMP) show a wide range where [PS]/[S] is linear with [PI, [PI
