The role for zinc in replication protein A.
Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly 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 presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-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.
Bochkareva
E
E
Department of Biochemistry and Molecular Biology, the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Korolev
S
S
Bochkarev
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051716:Replication Protein A; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004492:Edetic Acid; D014357:Trypsin; D015032:Zinc; D004229:Dithiothreitol