Sara K. Binz

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions.

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 double-stranded 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, RPA-mediated 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.

Cellular functions of human RPA1: Multiple roles of domains in replication, repair, and checkpoints

In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.

Functional Assays for Replication Protein A (RPA)

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures

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.

Lesion bypass by S. cerevisiae Pol ζ alone

DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

Regulatory Functions of the N-terminal Domain of the 70-kDa Subunit of Replication Protein A (RPA)

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes. RPA is composed of three subunits of 70, 32, and 14 kDa. The N-terminal domain of the 70-kDa subunit (RPA70) has weak DNA binding activity, interacts with proteins, and is involved in cellular DNA damage response. To define the mechanism by which this domain regulates RPA function, we analyzed the function of RPA forms containing a deletion of the N terminus of RPA70 and mutations in the phosphorylation domain of RPA (N-terminal 40 amino acids of the 32-kDa subunit). Although each individual mutation has only modest effects on RPA activity, a form combining both phosphorylation mimetic mutations and a deletion of the N-terminal domain of RPA70 was found to have dramatically altered activity. This combined mutant was defective in binding to short single-stranded DNA oligonucleotides and had altered interactions with proteins that bind to the DNA-binding core of RPA70. These results indicate that in the absence of the N-terminal domain of RPA70, a negatively charged phosphorylation domain disrupts the activity of the core DNA-binding domain of RPA. We conclude that the N-terminal domain of RPA70 functions by interacting with the phosphorylation domain of the 32-kDa subunit and blocking undesirable interactions with the core DNA-binding domain of RPA. These studies indicate that RPA conformation is important for regulating RPA-DNA and RPA-protein interactions.

Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties

Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability.