though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124 -15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reductionoxidation (redox) through its zinc finger domain. RPAssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.The replication protein A (RPA 1 ; also known as human single-stranded DNA-binding protein) is a three-subunit complex (70-, 34-, and 11-kDa; p70, p34, and p11, respectively) essential for DNA replication, nucleotide excision repair, and genetic recombination (54). In simian virus 40 (SV40) replication, RPA mediates unwinding of replication origin in the presence of SV40 T-antigen and topoisomerase I/II. During replication, it interacts with SV40 T-antigen and DNA polymerase ␣-primase (pol ␣-primase) complex (6, 7), which is necessary for the initiation of SV40 DNA replication (7-9). RPA is also involved in the elongation phase of DNA replication, because it stimulates pol ␣, pol ␦, and pol ⑀ activity on a primed template DNA (10, 11).In nucleotide excision repair, RPA interacts with several key repair proteins, Xeroderma pigmentosum (XP) group A-complementing protein, XPA (12-15), XPG (13), and XPF-excision repair cross-complementation group 1 (16). RPA stabilizes the XPA-damaged DNA complex through the interaction with XPA, which appears to be essential for DNA repair (14, 17). RPA itself can interact with UV-damaged DNA (18); however, the physiological relevance of RPA-damaged DNA interaction in DNA repair is not clear. RPA is also involved in the later stage of nucleotide excision repair, gap-filling reaction, in collaboration with proliferating cell nuclear antigen, replication factor-C, and pol ␦ (or pol ⑀) (19). In homologous recombination, RPA physically interacts with Rad51 and Rad52, which appears to be essential for initiation of recombination (20 -24).The large subunit of RPA, p70, has multiple functional domains, including pol ␣ stimulation, ssDNA binding, and a conserved zinc finger domain with 4-cysteine type (3,5,25). The ssDNA binding domain of RP...

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G 1 /S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs Cells exposed to UV irradiation are predominantly arrested in S-phase rather than at the G 1 /S boundary while repair occurs (1). The molecular mechanism of damage-induced Sphase arrest is not known; however, the effects of UV irradiation during S-phase on subsequent cell cycles are magnified in repair-deficient cells (2), indicating that these effects may be initiated by DNA damage itself. In contrast, in vitro replication experiments with cytosolic extracts from UV-damaged cells strongly indicate that UV-induced inhibition of replication is not due to a blockade of replication by DNA damage itself; rather, irradiation probably induces a mechanism that inhibits DNA replication (3, 4). It is not known how DNA damage induces the inhibition of DNA replication and yet permits DNA repair; however, proteins such as replication protein A (RPA 1 ; also known as human single-stranded DNA-binding protein) and proliferating cell nuclear antigen (PCNA) are involved in both processes (5-10) and may play a role in differential regulation. Earlier in vitro studies suggested that PCNA interacts with UV-induced protein, p21Cip1/Waf1 , which inhibits PCNA's function in DNA replication but not in repair (11-13). PCNA also interacts with GADD45 and MyD118, which are induced upon growth arrest and DNA damage, supporting a role for PCNA in damage-induced cell cycle arrest (14,15).RPA is a heterotrimeric single-stranded DNA-binding protein (70-, 34-, and 11-kDa subunits) originally identified as an essential factor for the replication of SV40 DNA (6, 9, 10). In addition to its role in replication, RPA is also required for DNA repair (5,16,17) and genetic recombination (18 -20), suggesting a possible role in regulation. In replication, RPA interacts with SV40 T-antigen and DNA polymerase ␣-primase complex, which probably mediates unwinding of SV40 origin-containing DNA (21-29). In addition, RPA stimulates polymerase ␣, ␦, and ⑀, which suggests its potential role in the elongation stage (30, 31). The middle subunit of RPA is phosphorylated in a cell cycle-dependent manner (32) and also by UV and ionizing radiation (3, 33). DNA-PK is responsible for the hyperphosphorylation of the 34-kDa subunit of RPA (34, 35); however, the in vivo observations with yeast and mammalian systems suggest additional involvement of other kinases, such as ataxia-telangiectasia mutant (ATM) (36,37). The observation that damageinduced RPA phosphorylation interferes with its interaction with p53 and DNA-PK su...

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes : replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase α-stimulation domain and a singlestranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa % -CysXaa "$ -Cys-Xaa # -Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of