Summary Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks (DSBs) that activate the DNA damage checkpoint. In budding yeast, the ATM homolog Tel1 associates preferentially with short telomeres and promotes telomere addition. Here we show that the telomeric proteins Rif1 and Rif2 attenuate Tel1 recruitment to DNA ends through distinct mechanisms. Both Rif1 and Rif2 inhibit the localization of Tel1, but not the Mre11-Rad50-Xrs2 (MRX) complex, to adjacent DNA ends. Rif1 function is weaker at short telomeric repeats compared with Rif2 function, and is partly dependent on Rif2. Rif2 competes with Tel1 for binding to the C-terminus of Xrs2. Once Tel1 is delocalized, MRX does not associate efficiently with Rap1-covered DNA ends. These results reveal a mechanism by which telomeric DNA sequences mask DNA ends from Tel1 recognition for the regulation of telomere length.

Double-strand breaks (DSBs) in chromosomal DNA elicit a rapid signaling response through the ATM protein kinase. ATM corresponds to Tel1 in budding yeast. Here we show that the catalytic activity of Tel1 is altered by protein binding at DNA ends via the Mre11-Rad50-Xrs2 (MRX) complex. Like ATM, Tel1 is activated through interaction with the MRX complex and DNA ends. In vivo, Tel1 activation is enhanced in sae2⌬ or mre11-3 mutants after camptothecin treatment; both of these mutants are defective in the removal of topoisomerase I from DNA. In contrast, an sae2⌬ mutation does not stimulate Tel1 activation after expression of the EcoRI endonuclease, which generates "clean" DNA ends. In an in vitro system, tethering of Fab fragments to DNA ends inhibits MRX-mediated DNA end processing but enhances Tel1 activation. The mre11-3 mutation abolishes DNA end-processing activity but does not affect the ability to enhance Tel1 activation. These results support a model in which MRX controls Tel1 activation by recognizing protein-bound DNA ends. Double-strand DNA breaks (DSBs) are deleterious DNA lesions that threaten genomic integrity if not precisely repaired. DSBs are induced not only by exogenous DNA-damaging agents but also during physiological cellular processes such as meiosis, lymphoid differentiation, and DNA replication. All organisms respond to DSBs by promptly launching the DNA damage response, which consists of checkpoint signaling and DNA repair (22,82). Cells possess two principal pathways for DSB repair: homologous recombination (HR) and nonhomologous end joining (NHEJ) (21). NHEJ rejoins DNA ends in the absence of significant homology (11, 36), whereas HR rejoins DSBs using a homologous donor sequence as a template (30). The Mre11-Rad50-Nbs1 (MRN) complex, which corresponds to the Mre11-Rad50-Xrs2 (MRX) complex in budding yeast, plays a key role in both the HR and NHEJ pathways (13,20,58,78). An early step in HR involves the generation of single-stranded DNA (ssDNA), followed by invasion of the template strand and DNA synthesis. To create ssDNA tracts at DSB ends, the MRN/MRX complex collaborates with several factors, including Sae2/Ctp1/CtIP, Dna2 nuclease, Sgs1/BLM helicase, and Exo1 exonuclease (18,33,37,44,60,83). Studies of budding yeast have proposed the model in which MRX and Sae2 act on DSBs at an earlier step than Sgs1, Dna2, and Exo1 (18,44,83). MRN/MRX is involved not only in generating ssDNA tracts but also in removing DNAprotein cross-links from DNA ends. The topoisomerase-like protein Spo11 becomes covalently bound to the 5Ј end of the DNA during meiotic DSB formation (28). MRX/MRN and Sae2/Ctp1 are involved in the removal of Spo11/Rec12 from 5Ј ends in budding and fission yeasts (23,29,43,49,59). The fission yeast MRN complex contributes to the removal of topoisomerase II from 5Ј ends as well as to the removal of topoisomerase I (Top1) from 3Ј ends (24).The checkpoint response that is activated by DSBs depends on the phosphatidylinositol 3-kinase related protein kinases ATM and ATR (22,82). Wher...

The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.

SummaryProliferating cell nuclear antigen (PCNA) is a wellknown multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms. We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix. PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.

Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits localization of Mre11 complex to DNA ends. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres. The results suggest that Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.

Polyacrylamide gel electrophoresis of the solubilized protein of rice dwarf virus (RDV), purified from RDV-infected rice plants, showed the virus to be composed of seven structural polypeptides. The molecular weights of these were calculated to be 193,000; 152,000; 131,000; 110,000; 62,000; 46,000 and 45,000 daltons.These were designated I, II, III, IV, V, VI, and VII respectively. Chymotrypsin-treatment of the virus was not effective for removing the outer components of capsid, whereas centrifugation of the virus through preformed cesium chloride gradients separated the preparation into four zones, top (T), upper-middle(M1), lower-middle (M2), and bottom (B), the buoyant densities of these being 1.26, 1.39-1.42, 1.43, and 1.46g/ml respectively. Recentrifugation of M1 through the cesium chloride gradients yielded three zones, M1, M2, and B, indicating that the latter two resulted from M1 by the shearing effect of cesium chloride centrifugation.Electron microscopy and SDSpolyacrylamide gel electrophoresis of B showed complete loss of II and IV, and a marked decrease of VI and VII, indicating that VI and VII compose the outer capsomeres and II and IV are the amorphous polypeptides that surround the outer capsomeres. The inner core was found to contain I, III, and V, among these V seemed to be the internal protein associated with nucleic acid because this was readily released from virions by freeze-thawing together with nucleic acid.

Page 1961, Fig. 1A and C: In the Rad53 and Tel1 panels, which were generated from the same Western blot and autoradiography images, the splice lines were not properly indicated and images were mixed up. The correct versions are shown below (left, A; right, C). Note that identical images were used for the WT cell lanes. Page 1962, Fig. 2A: An incorrect Western blot image was inadvertently used. The correct version is shown below. Note that the Tel1-HA lanes are identical to the ones used in Fig. 1A and C. Page 1962, Fig. 2C: A Western blot image was inadvertently duplicated and presented. Since we could not locate the original image, a set of data directly relevant to the experiment is shown below. These changes do not affect the interpretation of the data and conclusions of the study.