Circular dichroism spectra and electrophoretic mobility shift assays show that human replication protein A binds and melts intramolecular G-quadruplex structures.
Noncanonical DNA structures such as G-quadruplexes might obstruct the binding of hRPA, compromising the accuracy of replication, and be a source of genomic instability. In this study, circular dichroism (CD) and electrophoretic mobility shift assay (EMSA) experiments were used to show that hRPA can bind and melt nontelomeric, intramolecular DNA G-quadruplexes under physiologically germane conditions. EMSA results show that hRPA binds to a 58-mer that includes an embedded quadruplex with an affinity equal to or greater than to nonquadruplex forming 58-mers. Moreover, hRPA binds to a 26-mer purine-rich quadruplex-forming sequence with an affinity indistinguishable from that for binding to the complementary pyrimidine-rich sequence. Under the same conditions, hRPA does not have significant affinity for binding to the duplex formed from the two sequences. Thus, DNA secondary structures can significantly modulate the binding affinity of hRPA over and above its known preference for pyrimidine-rich single-stranded sequences, so that at least some intramolecular G-quadruplex structures may not inhibit hRPA binding during DNA replication. CD spectral changes in combination with EMSA titrations suggest that one hRPA heterotrimer is sufficient to form a stable complex with an unfolded 26-mer G-quadruplex prior to the binding of a second hRPA molecule.
Fan
Jun-Huei
JH
Department of Molecular and Cell Biology, Mail Stop FO31, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, USA.
Bochkareva
Elena
E
Bochkarev
Alexey
A
Gray
Donald M
DM
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D024202:Electrophoretic Mobility Shift Assay; D054856:G-Quadruplexes; D006358:Hot Temperature; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D055550:Protein Stability; D051716:Replication Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D051716:Replication Protein A