Replication protein A (RPA), a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA-) binding domains (DBDs) A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties. 1. Introduction Heterotrimeric replication protein A (RPA) is the primary eukaryotic single-stranded DNA- (ssDNA-) binding protein [1–3]. The three subunits are named RPA1 (70？kDa), RPA2 (32？kDa), and RPA3 (14？kDa) (Figure 1). RPA is a central player in all aspects of DNA metabolism, and it is thought to have little sequence specificity. RPA is a modular protein composed of several domains connected by flexible linkers, and it undergoes a conformational change upon ssDNA binding [4]. RPA is thought to assume a variety of structures depending on the nature of the DNA substrate [5]. This paper seeks to understand if RPA and its individual DNA-binding domains (DBDs) can selectively recognize any unique DNA sequences. Figure 1: RPA domain structure, constructs used in these studies and their corresponding crystal structures. RPA-AB contains RPA1 181-441. RPA-CDE includes RPA1 382-616 and intact RPA2 and RPA3. RPA-CDE-core consists of RPA1 438-616, RPA2 43-171, and intact RPA3. RPA-DE includes RPA2 43-171 and intact RPA3. RPA-C is composed of RPA1 432-595 with 7 point mutations (V435T, W442Q, V465T, V469T, F523S, F567S, and I571T). Crystal structure representations of RPA-AB apo, RPA-AB + dC 8, RPA-CDE-core, and RPA-DE are shown next to their domain maps [ 6– 9]. RPA binds ssDNA with high affinity ( ？M？1) and low cooperativity and binds polypyrimidine sequences with higher affinity than polypurine sequences [1, 13–15]. RPA contains six oligonucleotide binding (OB) folds (named A-F), five of which have previously been shown to possess DNA-binding activity (A-E) [1, 16]
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