Interaction of Spermatid-specific Protein TP2 with Nucleic Acids, in Vitro

TP2 was purified from rat testes employing a gentle method involving differential salt extraction of the sonication-resistant spermatid nuclei. The nucleic acid binding properties of TP2 were studied by fluorescence quenching, thermal denaturation, circular dichroism techniques and compared with those of TPl (Singh, J., and Rao, M. R. S. (1987) J. Biol. Chem. 262, 734740). The tyrosine fluorescence of TP2 was quenched upon binding to double-stranded and denatured DNA and poly(rA). The apparent association constants for binding of TP2 to these nucleic acids were calculated from the fluorescence quenching data, obtained at 60 mM NaCl, and found to be 1.63 x 10’ M-l, 6.5 X 10’ M-l, and 7.3 X 10’ M-l, respectively. Thermal denaturation studies of calf thymus DNA and its complexes with TP2 showed that at 1 mM NaCl, TP2 shifted the T,,, from 53 “C to 62-67 “C, while at 50 mM NaCl, the T, was shifted from 72 to 78 “C suggesting that TP2 is a DNA stabilizing protein. Circular dichroism studies of TPl *DNA and TP2*DNA complexes have revealed that TP2 has a better DNA condensing property than TPl. Furthermore, in contrast to TPl, TP2 does not destabilize in vitro the compactness of liver nucleosome core particles. The DNA binding properties of TPl and TP2 have been discussed in relation to the significance of their transient appearance during mammalian spermiogenesis.