Human replication protein (RPA) functions in DNA replication, homologous recombination and nucleotide excision repair. This multisubunit single-stranded DNA-binding protein may be required to make unique protein-protein contacts because heterologous single-stranded binding proteins cannot substitute for RPA in these diverse DNA transactions. We report here that, by using affinity chromatography and immunoprecipitation, we found that human RPA bound specifically and directly to two excision repair proteins, the xeroderma pigmentosum damage-recognition protein XPA (refs 8, 9) and the endonuclease XPG (refs 10-13). Although it had been suggested that RPA might function before the DNA synthesis repair stage, our finding that a complex of RPA and XPA showed a striking cooperativity in binding to DNA lesions indicates that RPA may function at the very earliest stage of excision repair. In addition, by binding XPG, RPA may target this endonuclease to damaged DNA.

Many recent reviews of eukaryotic DNA replication have emphasized our current understanding of either the initiation and regulation of eukaryotic nuclear DNA replication (1, 2), the DNA polymerases and other proteins involved (3-11), or the entire range of knowledge of the replication process (12)(13)(14)(15)(16)(17). Here, we will focus on recent findings concerning specific enzymatic reactions necessary for the growth of the eukaryotic replication fork. Synthesis of the Leading StrandThe separation of parental DNA strands determines the direction of movement of the replication fork. Because of the antiparallel structure of DNA, one new DNA strand is synthesized continuously in the direction of fork movement and is designated the leading strand. The other, or lagging strand, grows in the direction away from fork movement. On this strand, short discontinuous segments of DNA, called Okazaki fragments, are synthesized from RNA primers. During replication, the RNA primers are removed, and each fragment is joined to complete lagging strand synthesis.Knowledge of the reactions of the leading strand is derived largely from studies in vitro of the replication of simian virus 40 (SV40) 1 (1, 5, 12), a circular dsDNA virus with a single origin of replication. The viral protein large T antigen binds the origin and utilizes a 3Ј-5Ј-helicase activity to separate the strands creating two replication forks. Unwinding of the origin by large T antigen is stimulated by the ssDNA-binding protein replication protein A (RPA) (18 -21). After unwinding, each leading strand is initiated by an RNA primer generated by the primase subunits of DNA pol ␣ (22). The polymerase subunit of DNA pol ␣ then adds a stretch of deoxyribonucleotides to the RNA primer. Next, replication factor C (RFC) initiates a reaction called polymerase switching (23,24). In an ATP-dependent process, RFC dissociates DNA pol ␣ and assembles proliferating cell nuclear antigen (PCNA) in the region of the primer terminus. PCNA is a homotrimer of 36-kDa subunits that form a toroid structure. The current model suggests that RFC transiently opens the toroid of PCNA and then allows PCNA to reclose, encircling the double helix adjacent to the primer terminus (25,26). Structural analysis indicates that the central opening of PCNA contains sufficient clearance that the toroid can slide freely (27). Then, DNA pol ␦ interacts with PCNA, which functions as a sliding clamp holding the polymerase on the primer terminus. The clamped DNA polymerase is highly processive, adding thousands of nucleotides without dissociating.Recent structural analysis of DNA pol III holoenzyme from the homologous Escherichia coli system indicates that the sliding clamp (␤), the clamp-loading subunit (␥), and the polymerizing subunit (␣) form a complex at the primer terminus. ␣ is at the 3Ј-end and immediately behind are the other subunits contacting the double-stranded region of the primer-template (28). By both analogy and preliminary experimental evidence, RFC is retained in the complex of PCNA and D...

Flap endonuclease 1 (FEN1)1 is a member of the RAD2 superfamily of nucleases that play a critical role in DNA replication and repair in prokaryotes and eukaryotes (1-5). Both biochemical and genetic studies support a role for FEN1 during these cellular processes. Reconstitution reactions in vitro with either calf or human FEN1 illustrate the need for this nuclease during Okazaki fragment processing (6, 7) and long patch base excision repair (8). In Saccharomyces cerevisiae, a null mutant of the FEN1 homolog (RAD27/RTH1) exhibits slow growth, methyl methanesulfonate sensitivity, and hyper-recombination (9). These phenotypes are consistent with participation of FEN1 in both DNA replication and repair.Analysis of the complex substrate specificity of FEN1 aids in understanding its biological function. As a 5Ј-exonuclease, FEN1 can cleave either DNA or RNA-DNA primers annealed to templates (10). The presence of a primer bound immediately upstream of the cleavage site may stimulate, inhibit, or not affect FEN1 cleavage depending on the local sequence (10). Remarkably, FEN1 cleaves substrates with an unannealed 5Ј-tail or flap structure more efficiently than exonuclease substrates (11). Cleavage of a flap structure occurs at or near the point where the flap is annealed to the template strand. A specific feature of the FEN1 cleavage mechanism is that the nuclease appears to track on the flap to reach the cleavage site (2, 12, 13). FEN1 cannot cleave bubble substrates, which have a 5Ј-region of the flap annealed to the template (14). Primers annealed to the flap (12, 13) or biotin⅐streptavidin complexes on the flap (12) inhibit cleavage.Crystal structures of FEN1 homologs including T5 exonuclease (15), T4 RNase H (16), Methanococcus jannaschii FEN1 (17, 18), and Pyrococcus furiosus FEN1 (19) reveal a helical arch or loop. This arch may be utilized by the nuclease to recognize the 5Ј-end of an unannealed flap and to track along the flap structure. Mutational analyses of the loop region in the M. jannaschii FEN1 indicate that this physical structure is critical for both the binding and cleaving of flap substrates (17).PCNA has been found to be a potent stimulator of FEN1 cleavage activity (13,20). PCNA is the processivity factor for DNA polymerases ␦ and ⑀ (21). It is a toroidal homotrimer that is assembled around double-stranded DNA to form a sliding clamp (22). Biochemical and genetic analyses have shown that FEN1 interacts with a hydrophobic cleft located on the front face of the PCNA toroid (13,20,23,24). Interaction of PCNA with both polymerases and FEN1 suggests that it recruits FEN1 to the protein complex at the replication fork in vivo (25). PCNA also interacts with the regulatory protein p21 Cip1 , which is induced when chromosomal DNA is damaged (26 -28). Because p21Cip1 uses the same binding site on PCNA as replication proteins, it has been postulated that binding of this regulatory protein inactivates the replication complex while the chromosomal DNA is being repaired (23,29,30).Reconstitution reactions have re...

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3-tail on the upstream primer adjacent to the 5-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.Pathways for DNA replication, recombination, and repair are all proposed to involve DNA flap intermediates (1-8). The creation and resolution of single-stranded flap structures is a preferred method for removal of damaged or mismatched nucleotides. DNA replication requires the synthesis and joining of discontinuous segments, or Okazaki fragments. Each fragment is initiated by an RNA primer that must be removed prior to joining. Synthesis from the upstream fragment is thought to displace the RNA primer and some adjacent DNA into a single-stranded flap (8 -11). Processing of the flap intermediate is carried out by the structure-specific flap endonuclease 1 or FEN1 1 (4).FEN1 is evolutionarily conserved, and it has intrinsic 5Ј-3Ј-exonuclease and endonuclease activities (4,5,(12)(13)(14). The exonuclease activity utilizes double-stranded DNA with a nick or gap, whereas the endonuclease activity requires a flap structure. In prokarya, the FEN1 homologue is the 5Ј-nuclease domain associated with DNA polymerase I; however, FEN1 exists as a separate polypeptide in eukarya, archaea, and some bacteriophages (15). The mechanism and substrate specificity of FEN1 have been studied by many groups (4, 15-23). The preferred substrate for endonucleolytic cleavage was defined as a nick-flap substrate in vitro. It consists of upstream and downstream primers annealed to a template in a manner that creates a nick. The downstream primer contains a single-stranded 5Ј-flap region (4, 24).Sever...

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