Human Xeroderma Pigmentosum Group A Protein Interacts with Human Replication Protein A and Inhibits DNA Replication

Abstract

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase ␣-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase ␣ activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA. Replication protein A (RPA;1 also known as human singlestranded DNA binding protein, or HSSB), is a eukaryotic single-stranded DNA binding protein that contains three tightly associated subunits of 70, 34, and 11 kDa (p70, p34, and p11, respectively) (1-3). It is required for DNA replication, nucleotide excision repair, and homologous recombination (1-6), suggesting that it has multiple functions in DNA metabolic processes. The p34 subunit of RPA is phosphorylated at the G 1 /S boundary and dephosphorylated during mitosis (7,8). This phosphorylation event can also be induced by DNA damage (9, 10). Since DNA damage induces the inhibition of replication, RPA and the phosphorylation of its p34 subunit may play a role in the regulation of DNA replication (10).During the initiation of simian virus 40 (SV40) DNA replication, RPA interacts with SV40 large tumor antigen (T-ag) and the DNA polymerase ␣-primase complex (pol ␣-primase) (11, 12), which appears to be essential for DNA unwinding (12). Human RPA cannot be replaced at the initiation of replication by RPA from other species, suggesting that the interaction of RPA with other replication proteins may be crucial in this process. After unwinding, RPA is believed to both stabilize the unwound DNA and stimulate DNA polymerase ␣ (pol ␣) and DNA polymerase ␦ (pol ␦) activities, as determined by the elongation of primed DNA templates (13).In nucleotide excision repair, the requirement for RPA can be bypassed by incising DNA with the E. coli UvrABC enzyme. This observation suggests that RPA is involved in an early stage of UV excision repair (14). Although the role of RPA in repair is not yet well defined, the protein complex cannot be replaced by RPA from ...

In vitro analysis of the zinc-finger motif in human replication protein A

“…ZFM1 with a mutation at Cys-487 (mutated to alanine) showed ssDNA-binding activity similar to that of wt-RPA ( Figure 6). The two distict bands seen in Figure 6 represent RPA-DNA complexes with different ratios of RPA : DNA [25,29]. In contrast, ZFM2 and ZFM4 showed 2-3-fold lower ssDNA-binding activity in the presence of 0.1 µg of RPA (lanes 2 and 5 compared with lanes 8 and 11 in Figure 6).…”

“…The SV40 replication origin-containing plasmid, pSV01∆EP, has been described previously [25]. UV-irradiated pBS (3 kbp ; 450 J\m#) and p5A (4.5 kbp) were prepared as described previously [36].…”

“…SV40 Tag was prepared as described by Lee et al [37]. The ammonium sulphate fraction (35-65 %) and the phosphocellulose\1.0 M NaCl fraction of HeLa cell cytosolic extracts were prepared as described [25,37]. Purified human DNA pol δ and replication factor C (RF-C) were gifts from Dr. J. Hurwitz (Sloan-Kettering Cancer Institute, New York, NY, U.S.A.).…”

“…RPA is involved in the early stage of NER by interacting with XPA on damaged DNA, which allows the stable RPA-XPA complex to form on damaged DNA [22][23][24][25]27]. Since the zincfinger mutant failed to support NER, we were interested in examining whether the mutation at the zinc-finger domain affects XPA-DNA interaction.…”

“…In nucleotide excision repair (NER), RPA forms a complex with Xeroderma pigmentosum group A complementing protein (XPA) [22][23][24][25], a protein that specifically recognizes UV damage [26]. RPA stimulates the interaction of XPA with DNA through an RPA-XPA complex at damaged DNA sites [23,24,27].…”