Binding properties of replication protein A from human and yeast cells.

Abstract

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70,32, and 14 kDa Characterization of the mechanism of chromosomal DNA replication is essential for understanding the process of cell growth. Eukaryotic chromosomes are very large and complex; therefore, the study of model systems has been vital to reach our current understanding of DNA replication. One such model system, the papovavirus simian virus 40 (SV40), has properties very similar to those of cellular chromosomes (see recent reviews in references 7, 27, and 46). The SV40 genome exists in the cell in an authentic chromatin structure. Replication of SV40 DNA is dependent upon cellular replication proteins and a single virally encoded protein, large T antigen. SV40 DNA replication initiates at a specific DNA sequence and then proceeds bidirectionally, as does chromosomal replication. The development of a cell-free replication reaction capable of replicating SV40 DNA (34) led to the identification and purification of seven cellular proteins required for catalyzing DNA synthesis (36, 47, 52). Studies from several laboratories have led to a basic understanding of the mechanism of SV40 DNA replication (36, 47, 52). Initiation of replication occurs at a well-defined sequence, the SV40 origin of replication (Fig. 1)

RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

“…First, we measured the affinity of RPA for the 5′‐ssDNA flap region in the presence and absence of model leading strand and lagging strands using fluorescence anisotropy (Figs 3A and EV4B). In keeping with reported data on numerous other ssDNA‐containing substrates (Kim et al , 1992, 1994), we found the K D of RPA for the simple fork, but also the fork containing a model leading strand to be close to 6 nM (Fig 3A). Interestingly, the affinity for the model lagging‐strand substrate was also very similar to the simple model fork (K D  = 6.2 nM, Fig EV4B), indicating that the orientation/polarity of RPA, rather than simply the affinity of RPA on the fork substrate, might be critical for guiding incisions to the leading‐strand template.…”

“…To further establish the importance of the 5′‐ssDNA region, we generated substrates identical to those employed in Figs 1 and 2, except that the 5′‐ssDNA flap region was replaced by a 5′‐ssRNA flap (Fig 3B). The affinity of RPA for RNA is in the order of 10 −3 to 10 −4 fold less than for DNA (Kim et al , 1992). Consistent with the requirement for a 5′‐ssDNA flap for the stimulation of XPF‐ERCC1 by RPA, the RNA‐containing substrate was not able to stimulate XPF‐ERCC1 incision.…”

Mammalian base excision repair by DNA polymerases δ and ε

“…The presence of the single-stranded binding protein is even inhibitory for the long-patch repair reaction. RP-A combines with single-stranded DNA by forming a stable complex with at least 30 nucleotides (Kim et al, 1992). Since RP-A is required for nucleotide excision repair (Coverley et al, 1991) it appears that the gap size is critical in dictating whether this protein has a functional role in DNA repair.…”

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

“…The most common feature of RPA is its single-strand binding protein (SSB). In contrast to double-stranded DNA or RNA, RPA complex has shown a much higher binding affinity to single-stranded DNA (Kim et al, 1992). More than 25 years ago, RPA was firstly purified from human HeLa cell extracts in which RPA acts as a component of DNA replication for simian virus 40 (SV40) in vitro (Wobbe et al, 1987).…”