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The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial doublestranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPAmediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3-junction or a 5-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3-junctions was completely inhibited, thereby providing specificity to loading at 5-junctions. However, Rad17/3/1 loaded at 5-junctions can slide across double-stranded DNA to nearby 3-junctions and thereby affect the activity of proteins that act at 3-termini. These studies show a unique specificity of the checkpoint loader for 5-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.DNA damage elicits a broad range of cellular responses from DNA repair to inhibition of cell cycle progression. DNA damage checkpoints cause the arrest of cells at appropriate points in the cell cycle so that the integrity of the DNA can be restored (1). Although damage-induced checkpoints can be executed in many phases of the cell cycle, most of the progress in understanding the molecular details of this process has been made in the G 1 phase of the cell cycle, because its execution is unencumbered by complications related to the progression of DNA replication or chromosome segregation. In G 1 , for the DNA damage checkpoint to be executed in response to UV irradiation, repair of damage needs to be initiated by the nucleotide excision repair machinery (2). This finding suggests that gaps made during the progression of nucleotide excision repair are targets for the checkpoint machinery.Gaps in the double-stranded DNA contain three structural elements, a region of single-stranded DNA (ssDNA), 2 a ss-ds DNA junction with a 5Ј-terminus (5Ј-junction), and a ss-ds DNA junction with a 3Ј-terminus (3Ј-junction). Although the ssDNA is a target for binding by the single-stranded binding protein RPA, the 5Ј-and 3Ј-junction can be targeted by a variety of proteins, including nucleases (to either junction), DNA polymerases (to 3Ј-junctions), and circular clamp proteins. The replication clamp proliferating cell nuclear antigen (PCNA) is uniquely targeted to 3Ј-junctions and in that capacity serves as a processivity factor for several DNA polymerases and for a large number of other proteins that participate in various DNA metabolic pathways (reviewed in Ref.3). Much less is known about the PCNA-r...

No abstract

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.