BIOPOLYMERS
VOL. 15 (1976)
Reanalysis of Histone-Induced Conformational Changes in DNA Determined by Quasielastic Light Scattering
In a recent paper, Wun and Prins’ reported histone-induced changes in the translational diffusion coefficient ( 0 2 0 ) and terminal relaxation time (7int) of DNA as determined by quasielastic light scattering. Relaxation times faster than those of free DNA were reported for the DNA-histone F2A complex, which were ascribed to “supercoiling” in the double-helical structure. Since the opposite effect for the DNA-histone F1 complex was observed, i.e., slower relaxation times, the opposite phenomenon of “uncoiling” was proposed to explain that data. The results of Wun and Prins are summarized in Table I. It is shown in the present communication that the interpretation of “uncoiling” by histone F1 is not consistent with the known properties of DNA. Indeed, both “uncoiling” and “supercoiling” can result in faster times for translational diffusion and the terminal internal relaxation mode of DNA. Furthermore, the solvent conditions employed by these authors promote nucleoprotein dissociation; hence supercoiling would seem to be an improbable event. It is important in the ensuing argument to establish the relationship between the terminal internal relaxation time, the solvent viscosity ( q ) ,intrinsic viscosity ( [ q ] ) molecular , weight ( M r ) ,and absolute temperature ( T ) . According to Zimm’s formulation of the “bead-andspring” polymer,2 we might expect the proportionality
Assuming this relationship to be valid for the present system, then an approximate value of rlnt for the DNA of Wun and Prins (WP-DNA) can be computed from a known relaxation time for a different molecular weight DNA. The intrinsic viscosity for the WP-DNA is first estimated from the equation of Spatz and Crothers;’
M
= 3.82
x 104[~11.4
(2)
for M , = 1.5 X lo6,with the result [ q ] N 13.7 dl/g. Using the terminal orientational relaxation time T , ” ~= 0.018 sec for calf thymus DNA (M, = 15 X lo6, [ q ] = 72.5 dl/g) determined by zero angle depolarized light scattering a t Z5OC reported by Schmitz and Schurr4and neglecting solvent viscosity and temperature corrections, the expected relaxation time for the WP-DNA is, Tint =
(0.018)(13.7)(1.5X lo6) N 3.5 X (72.5)(15 X 106)
sec
sec.’ The discrepancy This value is comparable to Wun and Prins’ reported value of 5 X can be explained partially by the moderately large viscosity difference for the two solvents (0.02 M phosphate versus 0.8 M NaCl, 2 M urea). Nonetheless, the agreement is reasonable and it appears that the relationship in Eq. (1)is valid for the WP-DNA. There are two further consequences of the identification of the internal relaxation time for free DNA with the terminal relaxation time that are relevant to the interpretation of the histone F1-DNA system. First, the slower internal relaxation time reported for this system cannot be attributed to a slower internal mode of free DNA. Second, the internal relaxation time is directly proportional to the intrinsic viscosity. If uncoiling the DNA double-helical structure by histone F1 as proposed by Wun and Prins is tantamount to melting, thereby decreasing the intrinsic viscosity, then a faster internal relaxation time relative to free DNA should have been observed. The assignment of the terminal time to an intramolecular process is further suspect since histone F1 is known to dissociate from native chromatin in 0.5 M NaC1,5 which is below the salt concentration used by Wun and Prins. We suggest that some type of aggregation phenomenon, such as “temporal” intermolecular cross linking of neighboring DNA molecules by the histone F1 or self-aggregation of the F1 histones themselves, may be
2313 C 1976 by John Wiley & Sons, Inc.
BIOPOLYMERS VOL. 15 (1976)
2314
TABLE I Summary of t h e Results of Wun a n d Prins
a
b
Systema
D,, x 10’ (cm2/sec)
DNA~ histone F1-DNA histone F2A-DNA
1.6 1.0 2.6
x
lo4 (sec) 5 9.5 2.8
Solvent conditions: 0.8 M NaCl, 2 M urea, 20°C. M , = 1.5 X lo6.
responsible for the longer times reported for this system. The lysine-rich histones F16s7and F2C8 exist as oligomers in chromatin and nmr studies suggest histone F1 remains oligomeric after removal from the nucle~protein.~-” It seems equally untenable to invoke the concept of a “supercoiled DNA structure” for the histone F2A-DNA complex without additional supporting evidence. We are not aware of any studies in which histone F2A alone can induce conformational changes in DNA similar to that induced by the combination of all the histones of native chromatin (F2A1, F2A2, F3, and F2B). All four of these histones are required for the tightly coiled model of the monomeric unit (v-body, PS particle) proposed by Van Holde et al.13J4 Supercoiling associated with the tertiary structure of the nucleohistone complex would be unstable in 2 M urea as inferred by an increase in the reduced viscosity of native chromatin reported under these solvent conditions.12 Since the histones in chromatin can remain associated with the DNA even after a loosening of the supercoiled chromatin structure in 2 M urea,12histone F2A may cause “local uncoiling” of the DNA secondary structure giving rise to the reported faster relaxation times. We recognize that the above discussion is somewhat inconclusive because of the unusual solvent system employed by Wun and Prinz (0.8 M NaCl, 2 M urea), which makes it difficult to draw comparisons from existing literature on DNA-histone interactions. Our suggested mechanisms to account for the reported results should serve as possible explanations to be explored in better-defined systems. Hopefully, the present discussion may stimulate further quasielastic light-scattering investigations on the apparent unique role the lysine-rich histones have in chromatin condensation.
References 1. Wun, K. L. & P r i m W. (1975) Biopolymers 14,111-117. 2. Zimm, B. H. (1956) J . Chem. Phys. 24,269-278. 3. Spatz, H. Ch. & Crothers, D. M. (1969) J. Mol. Biol. 42,191-219. 4. Schmitz, K. S. & Schurr, J. M. (1973) Biopolymers 12,1543-1564. 5. Lake, R. S., Goidl, J. A. & Salzman, N. P. (1972) E r p . Cell. Res. 73,113-121. 6. Bonner, W. M. & Pollard, H. B. (1975) Biochem. Biophys. Res. Commun. 64, 282288. 7. Chalkley, R. (1975) Biochem. Eiophys. Res. Commun. 64,587-594. 8. Olins, 0. E. & Wright, E. B. (1973) J. Cell Bid. 59,304-317. 9. Bradbury, E. M. & Rattle, H. W. E. (1972) Eur. J. Biochem. 27,270-281. 10. Bradbury, E. M., Carpenter, B. G. & Rattle, H. W. E. (1973) Nature 241,123-126. 11. Bradbury, E. M., Danby, S. E., Rattle, H. W. E. & Giancotti, V. (1975)Eur. J. Biochem. 57,97-105. 12. Bartley, J. A. & Chalkley, R. (1968) Biochim. Biophys. Acta 160,224-228.
COMMUNICATIONS TO T H E EDITOR
2315
13. Van Holde, K. E., Sahasrabuddhe, C. G. L& Ramsay-Shaw, B. (1974) Nucl. Acids Res. 1, 1579-1586. 14. Ramsay-Shaw, B., Herman, T. K., Kovacic, R. T., Beaudreau, G. S. & Van Holde, K. E. (1976) Proc. N a t . Acad. Sci. U.S. 73,505-509.
BARBARA RAMSAY-SHAW Department of Chemistry Duke University Durham, North Carolina 27706 KENNETHS. SCHMITZ Department of Chemistry University of Missouri Kansas City, Missouri 64110 Received March 4,1976 Returned for revision May 24, 1976 Accepted June 21, 1976
VOL. 15 (1976)
Reanalysis of Histone-Induced Conformational Changes in DNA Determined by Quasielastic Light Scattering
In a recent paper, Wun and Prins’ reported histone-induced changes in the translational diffusion coefficient ( 0 2 0 ) and terminal relaxation time (7int) of DNA as determined by quasielastic light scattering. Relaxation times faster than those of free DNA were reported for the DNA-histone F2A complex, which were ascribed to “supercoiling” in the double-helical structure. Since the opposite effect for the DNA-histone F1 complex was observed, i.e., slower relaxation times, the opposite phenomenon of “uncoiling” was proposed to explain that data. The results of Wun and Prins are summarized in Table I. It is shown in the present communication that the interpretation of “uncoiling” by histone F1 is not consistent with the known properties of DNA. Indeed, both “uncoiling” and “supercoiling” can result in faster times for translational diffusion and the terminal internal relaxation mode of DNA. Furthermore, the solvent conditions employed by these authors promote nucleoprotein dissociation; hence supercoiling would seem to be an improbable event. It is important in the ensuing argument to establish the relationship between the terminal internal relaxation time, the solvent viscosity ( q ) ,intrinsic viscosity ( [ q ] ) molecular , weight ( M r ) ,and absolute temperature ( T ) . According to Zimm’s formulation of the “bead-andspring” polymer,2 we might expect the proportionality
Assuming this relationship to be valid for the present system, then an approximate value of rlnt for the DNA of Wun and Prins (WP-DNA) can be computed from a known relaxation time for a different molecular weight DNA. The intrinsic viscosity for the WP-DNA is first estimated from the equation of Spatz and Crothers;’
M
= 3.82
x 104[~11.4
(2)
for M , = 1.5 X lo6,with the result [ q ] N 13.7 dl/g. Using the terminal orientational relaxation time T , ” ~= 0.018 sec for calf thymus DNA (M, = 15 X lo6, [ q ] = 72.5 dl/g) determined by zero angle depolarized light scattering a t Z5OC reported by Schmitz and Schurr4and neglecting solvent viscosity and temperature corrections, the expected relaxation time for the WP-DNA is, Tint =
(0.018)(13.7)(1.5X lo6) N 3.5 X (72.5)(15 X 106)
sec
sec.’ The discrepancy This value is comparable to Wun and Prins’ reported value of 5 X can be explained partially by the moderately large viscosity difference for the two solvents (0.02 M phosphate versus 0.8 M NaCl, 2 M urea). Nonetheless, the agreement is reasonable and it appears that the relationship in Eq. (1)is valid for the WP-DNA. There are two further consequences of the identification of the internal relaxation time for free DNA with the terminal relaxation time that are relevant to the interpretation of the histone F1-DNA system. First, the slower internal relaxation time reported for this system cannot be attributed to a slower internal mode of free DNA. Second, the internal relaxation time is directly proportional to the intrinsic viscosity. If uncoiling the DNA double-helical structure by histone F1 as proposed by Wun and Prins is tantamount to melting, thereby decreasing the intrinsic viscosity, then a faster internal relaxation time relative to free DNA should have been observed. The assignment of the terminal time to an intramolecular process is further suspect since histone F1 is known to dissociate from native chromatin in 0.5 M NaC1,5 which is below the salt concentration used by Wun and Prins. We suggest that some type of aggregation phenomenon, such as “temporal” intermolecular cross linking of neighboring DNA molecules by the histone F1 or self-aggregation of the F1 histones themselves, may be
2313 C 1976 by John Wiley & Sons, Inc.
BIOPOLYMERS VOL. 15 (1976)
2314
TABLE I Summary of t h e Results of Wun a n d Prins
a
b
Systema
D,, x 10’ (cm2/sec)
DNA~ histone F1-DNA histone F2A-DNA
1.6 1.0 2.6
x
lo4 (sec) 5 9.5 2.8
Solvent conditions: 0.8 M NaCl, 2 M urea, 20°C. M , = 1.5 X lo6.
responsible for the longer times reported for this system. The lysine-rich histones F16s7and F2C8 exist as oligomers in chromatin and nmr studies suggest histone F1 remains oligomeric after removal from the nucle~protein.~-” It seems equally untenable to invoke the concept of a “supercoiled DNA structure” for the histone F2A-DNA complex without additional supporting evidence. We are not aware of any studies in which histone F2A alone can induce conformational changes in DNA similar to that induced by the combination of all the histones of native chromatin (F2A1, F2A2, F3, and F2B). All four of these histones are required for the tightly coiled model of the monomeric unit (v-body, PS particle) proposed by Van Holde et al.13J4 Supercoiling associated with the tertiary structure of the nucleohistone complex would be unstable in 2 M urea as inferred by an increase in the reduced viscosity of native chromatin reported under these solvent conditions.12 Since the histones in chromatin can remain associated with the DNA even after a loosening of the supercoiled chromatin structure in 2 M urea,12histone F2A may cause “local uncoiling” of the DNA secondary structure giving rise to the reported faster relaxation times. We recognize that the above discussion is somewhat inconclusive because of the unusual solvent system employed by Wun and Prinz (0.8 M NaCl, 2 M urea), which makes it difficult to draw comparisons from existing literature on DNA-histone interactions. Our suggested mechanisms to account for the reported results should serve as possible explanations to be explored in better-defined systems. Hopefully, the present discussion may stimulate further quasielastic light-scattering investigations on the apparent unique role the lysine-rich histones have in chromatin condensation.
References 1. Wun, K. L. & P r i m W. (1975) Biopolymers 14,111-117. 2. Zimm, B. H. (1956) J . Chem. Phys. 24,269-278. 3. Spatz, H. Ch. & Crothers, D. M. (1969) J. Mol. Biol. 42,191-219. 4. Schmitz, K. S. & Schurr, J. M. (1973) Biopolymers 12,1543-1564. 5. Lake, R. S., Goidl, J. A. & Salzman, N. P. (1972) E r p . Cell. Res. 73,113-121. 6. Bonner, W. M. & Pollard, H. B. (1975) Biochem. Biophys. Res. Commun. 64, 282288. 7. Chalkley, R. (1975) Biochem. Eiophys. Res. Commun. 64,587-594. 8. Olins, 0. E. & Wright, E. B. (1973) J. Cell Bid. 59,304-317. 9. Bradbury, E. M. & Rattle, H. W. E. (1972) Eur. J. Biochem. 27,270-281. 10. Bradbury, E. M., Carpenter, B. G. & Rattle, H. W. E. (1973) Nature 241,123-126. 11. Bradbury, E. M., Danby, S. E., Rattle, H. W. E. & Giancotti, V. (1975)Eur. J. Biochem. 57,97-105. 12. Bartley, J. A. & Chalkley, R. (1968) Biochim. Biophys. Acta 160,224-228.
COMMUNICATIONS TO T H E EDITOR
2315
13. Van Holde, K. E., Sahasrabuddhe, C. G. L& Ramsay-Shaw, B. (1974) Nucl. Acids Res. 1, 1579-1586. 14. Ramsay-Shaw, B., Herman, T. K., Kovacic, R. T., Beaudreau, G. S. & Van Holde, K. E. (1976) Proc. N a t . Acad. Sci. U.S. 73,505-509.
BARBARA RAMSAY-SHAW Department of Chemistry Duke University Durham, North Carolina 27706 KENNETHS. SCHMITZ Department of Chemistry University of Missouri Kansas City, Missouri 64110 Received March 4,1976 Returned for revision May 24, 1976 Accepted June 21, 1976
