BIOPOLYMERS
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`VOL. 13, 669-675 (1974)
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`The Effect of Thymine Dimers on
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`DNA:DNA Hybridization
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`MICHAEL KAHN, Department of Biological Sciences, Stanford University,
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`Stanford, California 94305
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`Synopsi
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`DNA from bacteriophage T7 was irradiated at long ultraviolet wavelengths in the pres-
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`ence of silver ions. Such treatment leads to selective production of thymine:thymine
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`dimers in DNA. The DNA was melted and the renaturation rate was determined as a
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`function of thymine dimer content and renaturation temperature. Under “normal”
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`hybridization conditions little change in the renaturation rate was observed even when
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`30% of the thymine was dimerized. This result is consistent with the view that up to a
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`15% change in the primary sequence of DNA does not appreciably change the renatura-
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`tion rate.
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`The kinetics of nucleic acid hybridization have been shown to be depen-
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`dent on a variety of factors such as temperature, ionic strength, and gua-
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`nine—cytosine content.‘ A long-standing question is to what extent a
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`mismatch of bases (i.e., unconventional pairing of bases) will affect the
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`renaturation rate.‘ Since the measurable parameter in the Wetmur and
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`Davidson formulation” of renaturation kinetics KN contains both a com-
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`plexity correction factor and a putative mismatch correction factor, the
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`estimation of complexity based solely on KN might be questioned in cases
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`in which mismatch is known to be present.“
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`As an example of the effect of mismatching, reannealed hybrids of the
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`rapidly renaturing fraction of eukaryotic DNA show a decreased melting
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`temperature after renaturation, indicating that the hybrid contains mis-
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`matched bases.5 Direct sequence analysis of the guinea pig oz satellite
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`DNA suggests that mutational change in the primary DNA sequence is
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`common and will
`lead to mismatch upon hybridization. Sutton and
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`McCallum4 reannealed mouse satellite DNA and subsequently separated
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`the duplexes into four classes on the basis of differing melting temperature
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`T,,,. The rate of renaturation of these four classes was strongly dependent
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`on their respective T,,,’s———an observation that led Southern to propose” that
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`the lowered thermal stability was the result of mutational changes in some
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`homogeneous primal sequence and that these changes reduced the rate of
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`hybrid formation.
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`The system described in this paper was designed to determine whether a
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`correction for mismatch should be applied to kinetic hybridization data.
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`In this system the intrastrand thymine dimer was used to model a muta-
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`© 1974 by John Wiley & Sons, Inc.
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`Exhibit 21 15 Page 1
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`Enzo Exhibit 21 15
`BD v. Enzo
`Case |PR2017-00181
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`Enzo Exhibit 2115
`BD v. Enzo
`Case IPR2017-00181
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`Exhibit 2115 Page 1
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`KAHN
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`tional mismatch. The choice was justified by the knowledge that a dimer
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`decreases the stability of the DNA duplex and that its action is at the level
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`of primary structure. These are the minimum defining properties of mis-
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`match. Brunk“ and Shafranovkaya et a1." concluded by different methods
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`that at low dimer concentrations the distribution of dimers in DNA is not
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`random. Brunk showed that the longer pyrimidine tracts contained a
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`greater than expected percentage of thymine as dimers. At a high level
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`of dimerization the dimers are as random as the pyrimidine tracts in which
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`they occur.“
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`The advantages of the thymine dimer as a specific lesion in DNA are
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`numerous. Using silver ions, high yields of dimers are possible with
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`negligible contribution from other photoproductsf’ Double—stranded
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`DNA can be used and therefore melting temperatures will show no effects
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`due to self-sorting of altered strands.
`Introduction of the photoproduct
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`is rapid and convenient, and the photoproducts have been Well charac-
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`terized and are fairly simple to assay.
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`Similar model systems have been used in attempts to determine the
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`effect of mismatch on renaturation rate. Deamination has been used in
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`order to model transition-type mutations.9‘“ Transversions have been
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`modeled by using glyoxal to complete a third ring on guanosine residues”
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`and by using chloroacetaldehyde to modify adenine and cytosine.”
`In all
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`of these studies renaturation rate was not found to be very dependent on
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`the presence of the alteration; the maximum rate depression reported is 80%
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`for heavily glyoxylated DNA with a melting temperature 24°C lower than
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`that of native DNA. “
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`MATERIALS AND METHODS
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`DNA
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`T7 wild-type phage was obtained from M. Chamberlain; E. coli strain
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`B/r thy‘ was obtained from H. Nakayama. Unlabeled T7 DNA was
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`prepared in a manner similar to that of England.” Tritium labeled DNA
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`was prepared by growing E. coli B/r thy‘ in 0.2% glucose, 0.1% casamino
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`acids (Difeo) to an OD of 0.8 in a medium containing 3H (methyl) thymine
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`(1 pg/ml, 2 pm Ci/ml NEN) and infecting with T7 at a multiplicity of 0.1.
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`Phage were prepared following Thomas and Abelson.” All phage were
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`sedimented in CsCl step gradients and banded at equilibrium. The DNA
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`was extracted with phenol, dialyzed against 0.06 M NaH2PO4, 0.06 M
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`N-a;HP04,
`(0.12 M NaP), and stored over chloroform. Concentration
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`was measured by determining A250.
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`Formation of Dimers
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`DNA was irradiated at 7 or 70 pg DNA/ ml for Various lengths of time
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`with a high-pressure mercury lamp (PEK 10010) through a 1.6-mm glass
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`filter. The glass had a transmittance of less than 2% at 300 my and 47%
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`Exhibit 21 15 Page 2
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`Exhibit 2115 Page 2
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`DNA: DNA HYBRIDIZATION
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`at 310 mp. Silver nitrate was added to a Ag:DNA-phosphate ratio of
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`greater than 1. Water-saturated nitrogen was bubbled through the DNA—
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`silver solution prior to and during irradiation. An aliquot of the irradiated
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`DNA was acid hydrolyzed and analyzed for photoproducts on Dowex
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`formate columns and by paper chromatography. 15
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`Hybridization
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`The irradiated DNA was dialyzed once against 0.02 M NaCN and then
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`twice against 0.12 M NaP to remove Ag+. Release of silver was com-
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`plete as judged by disappearance of Ag“°"‘ in control experiments. The
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`DNA was then sonicated and its molecular weight determined by sedi-
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`mentation through a 5—20% alkaline sucrose gradient using a method
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`similar to that of Abelson and Thomas” (Spineo 50.1 rotor, 46,000 rpm,
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`6 hr, 20°C). Molecular weights were in the range of 3~5 X 105 daltons.
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`DNA samples were sonicated (Bronson LS—75 sonifier) and melted at
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`100°C for 10 min, then quick cooled in ice water. The solutions were
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`transferred to shell vials mounted in a floating shell vial holder and placed
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`in a constant temperature bath.; 0.1-ml samples were taken at predeter-
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`mined times and placed in 1 ml of ice-cold 0.12 M NaP. These were stored
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`cold until analyzed for duplex by a hydroxylapatite—centrifuge method
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`similar to that of Brenner et al.” The rate constant K was determined and
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`corrected for length and concentration effects.”
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`Melting Temperature
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`Sonicated DNA was dialyzed against 0.01 M NaCl, 0.001 M Na;H2EDTA,
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`0.003 M NaP pH = 7, and placed in quartz cuvettes. Temperature was
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`raised using a temperature-controlled euvette holder connected to a Haake
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`circulating water bath. Absorbance at 260 nm was measured with a Zeiss
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`PMQ, II spectrophotometer. Temperature regulation was accurate to
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`0.2°C (R. Baldwin, personal communication) and DNA standards were
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`melted at the same time. Change in melting temperature AT,,, was taken
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`to be the temperature difference between the midpoints of the hyper-
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`chromicity curve of the sample and that of the standard.
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`RESULTS AND DISCUSSION
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`The specificity of the silver—sensitization technique for dimer formation
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`was examined. Acid stable nondimer photoproducts contributed less than
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`0.1% of total label as determined by column chromatography.“ The
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`contribution of cytosine dimers (CT) was less than 1% of the total label,
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`u11der conditions in which 30% of the label was identified as dimer as-
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`sociated. Strand breakage was observed but only with unsonicated DNA
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`and times of irradiation longer than those used in this study. Sonication
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`after irradiation eliminated strand breakages as a factor in the actual
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`renaturation. No evidence for an increased extent of strand breakage was
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`observed in dimerized DNA at higher temperatures.
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`Exhibit 21 15 Page 3
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`Exhibit 2115 Page 3
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`672
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`KAHN
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`Figure 1 is a plot of total dimers (mostly thymine dimers) versus ATM.
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`Regression of the points leads to the empirical formula
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`ATM = Klfil/T
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`where K = 06°C per dimer per 100 base pairs (°C/%). Such a figure is
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`lower than the value of K = 12° C/% obtained by Hutton and Wetmur“
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`for glyoxylated DNA and 1.6°C/% obtained by Ullman and McCarthy for
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`deaminated DNA.“ A single dimer is thus approximately half as destabi-
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`lizing as these other alterations.
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`Irradiated DNA hydro-
`Fig. 1. Effect of thymine dimers on melting temperature.
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`lyzed by heating at 121°C for 3 hr in equal volume of HCIO4. Solution then neutralized
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`with KOH and solid KCIO4 removed. Sample applied to column of AG 1-X8 resin
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`(formate form, BioRad), which had been pre-equilibrated with 0.02 M NH.0H. Dimers
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`eluted with 0.02 M NH.OH, 0.02 M HCOOH pH = 8, monomers with 002 M HCOOH.
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`Plotted are dimer counts per total counts. AT". determined as described in “Methods.”
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`Fig. 2. Melting profiles of irradiated DNA. Dimer concentration 29.0% (A), 10% (O),
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`6.8% (O), or 0% (A).
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`Exhibit 21 15 Page 4
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`Exhibit 2115 Page 4
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`DNA: DNA HYBRIDIZATION
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`The width of the melting curves indicates a sharp transition (Figure 2).
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`This contrasts with the broad melt of DNA irradiated at A = 254 mp but is
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`consistent with results obtained by irradiating at >\ = 310 my in the pres-
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`ence of acetophenone.”
`It is probable that the former results are due to a
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`variety of photoproducts other than dimers, which have greater destabiliz-
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`ing effects than dimers. These photoproducts are absent in the DNA
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`used in the present work.
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`Renaturation Rate
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`Sonicated T7 DNA was reannealed as described in “Methods” and the
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`Cot. curves plotted in Figure 3. The minimum rate observed at 65°C is
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`62% of that for unaltered DNA. This change, with DNA containing 29%
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`of its thymine label as dimers and having a AT,,, of 10.2°C indicates that in a
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`stability region comparable to reannealed eukaryotic DNA, the rate con-
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`stant is not altered by even a factor of 2.
`. The extent of dimerization is not
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`extensive enough to test the hypothesis of Hutton and Wetmur“ concern-
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`ing the precise form of the dependence of rate constant on AT,,, but is
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`consistent with their data in the range explored in this study.
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`In order to see how the rate constant varies as the conditions of hybridiza-
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`tion were made more stringent, the incubation temperature was raised.
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`Results are plotted in Figure 4. The dimer—dependent decrease in renatura-
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`tion rate becomes more pronounced as temperature rises.
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`0.5
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`REASSOCIATED l.0
`FRACTION
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`I0‘3
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`no’?
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`10
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`C,t
`(moles/sec/I)
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`Fig. 3. Reassociation of irradiated DNA. Samples obtained as described in text were
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`added to 0.5 g hydroxylapatite (DNA grade, BioRad) suspended in 10 ml of 0.12 M NaP;
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`after equilibration at 65°C samples centrifuged in Sorvall desk-top centrifuge. Single-
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`stranded DNA in supernatant precipitated in 5% cold Cl3CCOOH. Pellet resuspended
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`in 0.4 M NaP, centrifuged, and supernatant double-stranded DNA precipitated. Sam-
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`ples filtered through Millipore filters, and counted in toluene-PPO-POPOP. Plotted is
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`ratio SS/(SS + DS). Dimer content 29.0% (A), 22.6% (I), 10.0% (0) and 0% (A).
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`Exhibit 21 15 Page 5
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`Exhibit 2115 Page 5
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`

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`674
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`KAHN
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` 0.2 0.3
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`0.4
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`f‘
`'r,T
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`(K/K0) is rate
`Fig. 4. Effect of annealing temperature on rate of reassociation.
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`constant at given dimer concentration divided by rate constant of unirradiated DNA.
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`Renaturations performed at 65°C (0), 75°C (0), 80°C (A) and 85°C ([3).
`29% dimer-
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`ized sample melts at 84.5°C in this buffer (0.12 NaP) as determined by hydroxyapatite
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`binding.
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`A correction to the renaturation constant is needed but this correction
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`is sma1l-—3.5%/ °C of AT,,, at 65°C. Such a factor is not large enough to
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`account for the results of Sutton and McCallum.‘ This study therefore
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`supports the hypothesis of Hutton and Wetmur,“ that the rate difference
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`is due to classes of differing complexities and thermal stabilities, rather
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`than Southern’s random mutation hypothesis.“
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`The author thanks Dr. Philip Hanawalt for timely advice and encouragement. This
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`work was supported by Atomic Energy Commission Grant AT(04-3)326-7 to Philip
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`Hanawalt and a National Institutes of Health predoctoral traineeship to the author.
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`References
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`Walker, P. M. B. (1969) Proyr. Nucleic Acid Res. Mol. Biol. 9, 301-326.
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`Wetmur, J. G. & Davidson, N. (1968) J. Mol. Biol., 31, 349-370.
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`Southern, E. M. (1971) Nature New Biol., 232, 82-83.
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`Sutton, W. D. & McCallum, M. (1971) Nature New Biol., 232, 83-85.
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`Britten, R. J. & Kohne, D. E. (1968) Science 161, 529-540.
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`Brunk, C. F. (1973) Nature New Biol. 241, 74-76.
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`. Shafranovkaya, N. N., Trifonov, E. N., Lazurkin, Yu. S. & Frank-Kamenetskii,
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`M. D. (1973) Nature New Biol. 241 58-60.
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`8. Rahn, R. 0. & Landry, L. C. Photoclem. Photobiol. (submitted).
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`9. McCarthy, B. J. & Farquhar, M. N. (1972) in Evolution of Genetic Systems, H. H.
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`Smith, Ed., Brookhaven, Symp., 23 pp. 1-43.
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`10. Bonner, T., Brenner, D. & Britten, R. (1971) Carnegie Inst. Washington Yearbook,
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`71. 287-289.
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`11. Hutton, J. R. & Wetmur, J. G. (1973) Biochemistry 12, 558-563.
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`12. Lee, C. H. & Wetmur, J. G. (1973) Biochem. Biophys. Res. Commun. 50, 879-
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`885.
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`13. England, P. T. (1972) J. Mol. Biol. 66, 209-224.
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`14. Thomas, C. A. & Abelson, J. (1966) in Procedures in Nucleic Acid Research, G. L.
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`Cantoni & D. R. Davies, Eds., V, l , 553-568.
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`".°°S"t"9-".l°!"
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`Exhibit 21 15 Page 6
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`Exhibit 2115 Page 6
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`DNA: DNA HYBRIDIZATION
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`675
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`15. Lamola, A. A. (1969) Photochem. Photobiol. 9, 291-294.
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`16. Abelson, J. & Thomas, C. A. (1966) J. Mol. Biol. 18, 262-291.
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`17. Brenner, D. J., Fanning, G. R., Rake, S. V. & Johnson, K.E. (1969) Anal. Bio-
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`chem. 28, 447-459.
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`18. Ullman, J. & McCarthy, B. J. (1973) Biochim. Biophys. Acta 294, 405-424.
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`19. Charlier, M., Helene, C. & Carrier, W. L. (1972) Photochem. Photobiol. 15, 527-
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`536.
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`Received September 24, 1973
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`Revised January 2, 1974
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`Exhibit 21 15 Page 7
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`Exhibit 2115 Page 7
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