The role for zinc in replication protein A

Heterotrimeric human single stranded DNA (ssDNA)binding protein, replicatio n protein A (RPA), is a central player in DNA replication, recombination, a nd repair. The C terminus of the largest subunit, RPA70, contains a putativ e zinc-binding motif and is implicated in complex formation with two smalle r subunits, RPA14 and RPA32, The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectrosco py. The proteolytic core of this domain comprised amino acids 432-616, X-ra y fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(I I). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (a mino acids 43-171), and RPA14 had strong DNA binding activity. When properl y coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the pre sence of chelating agents. Our data indicate that (i) Zn(II) is essential t o stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses D NA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.