No abstract

The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17͞3͞1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17͞3͞1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17͞3͞1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17͞3͞1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17͞3͞1 showed no detectable exonuclease activity. D NA damage in eukaryotic cells provokes a range of cellular responses which includes DNA repair, apoptosis, and cellcycle arrest. The DNA damage checkpoint response arrests cells at appropriate points in the cell cycle to allow recovery of the integrity of the DNA before reentering the cell cycle (1). The later steps of the DNA damage checkpoint that ultimately lead to inhibition of the cdk kinases that drive the cell cycle are relatively well understood (recently reviewed in refs. 2 and 3). However, molecular details about the initial steps of damage recognition that activate the checkpoint response are still lacking. Two distinct complexes independently localize to sites of DNA damage in Saccharomyces cerevisiae, but the presence of both complexes is required for proper checkpoint function (4-6). The Mec1 protein kinase forms a complex with Ddc2 and may function in both DNA damage recognition and signal transduction (6, 7). Another set of proteins, Rad24, Rad17, Mec3, and Ddc1, which may primarily function in DNA damage recognition and processing, shows sequence similarities with replication factor C (RFC) and proliferating cell nuclear antigen (PCNA), the eukaryotic clamp loader-clamp system that is central to the structure of the replication fork. RFC is a heteropentameric complex, consisting of a large subunit, Rfc1, and the four small Rfc2-5 subunits, that clamps PCNA around the DNA at primer͞template junctions in an ATP-dependent process (8). PCNA is the processivity factor of DNA polymerase ␦ and many other proteins involved in various aspects of DNA metabolism (9).Rad24 (Rad17 in human and Schizosaccharomyces pombe) shows sequence similarity with the Rfc1 subunit of RFC and interacts with the small subunits of RFC (10-12). A heteropentameric complex, consisting of Rad24 and the Rfc2-5 subunits, has been purified from yeast, and the analogous human complex has been purified from ov...

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...

Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.

DNA polymerase (Pol ), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol , yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol . Efficient PCNA stimulation required that PCNA was prevented from sliding off the damagecontaining model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase ␦ or (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damageinduced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol or by DNA polymerase ␦.During the S phase of the eukaryotic cell cycle, when DNA damage may form a block for the high fidelity DNA polymerases such as DNA polymerase ␦ (Pol ␦) 1 or Pol ⑀, several complex pathways are activated. The Saccharomyces cerevisiae RAD6 and RAD18 genes control a bifurcating pathway that activates translesion synthesis (TLS) by error-prone DNA polymerases as well as a recombinational damage avoidance pathway (reviewed in Ref. 1). Pol is largely responsible for the relatively error-free bypass of cis-syn pyrimidine dimers in the template strand (2, 3). In contrast, most other types of damage are bypassed by a set of error-prone DNA polymerases, and this bypass forms the molecular basis for damage-induced mutagenesis in the cell. One of these translesion polymerases is Pol , a heterodimer of the Rev3 and Rev7 subunits, and the second enzyme is the Rev1 deoxycytidyl transferase (4, 5). The replicative polymerase Pol ␦ has also been shown to be required for damage-induced mutagenesis, primarily through mutational studies of its Pol32 subunit (3, 6 -8). Consequently, a model has evolved in which Pol ␦ inserts a nucleotide across template damage, whereas Pol and Rev1 are responsible for extension of that inserted nucleotide. In analogy to bacterial mutagenesis systems, this model has been named the twopolymerase model for TLS (9, 10). Biochemically, this model has mainly been studied by measuring bypass rates and efficiencies of the catalytic cores of these enzymes without the inclusion of processivity factors such as the proliferating cell nuclear antigen (PCNA) (11,12).Most DNA polymerases possess a unique consensus motif, QxxLxxFF, that is required for binding of the polymerase to PCNA and for PCNA-mediated processive DNA synthesis (reviewed in (13)). For ...

No abstract

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

No abstract