Sister chromatid cohesion role for CDC28-CDK in Saccharomyces cerevisiae

High-fidelity chromosome segregation requires that the sister chromatids produced during S phase also become paired during S phase. Ctf7p (Eco1p) is required to establish sister chromatid pairing specifically during DNA replication. However, Ctf7p also becomes active during G(2)/M in response to DNA damage. Ctf7p is a phosphoprotein and an in vitro target of Cdc28p cyclin-dependent kinase (CDK), suggesting one possible mechanism for regulating the essential function of Ctf7p. Here, we report a novel synthetic lethal interaction between ctf7 and cdc28. However, neither elevated CDC28 levels nor CDC28 Cak1p-bypass alleles rescue ctf7 cell phenotypes. Moreover, cells expressing Ctf7p mutated at all full- and partial-consensus CDK-phosphorylation sites exhibit robust cell growth. These and other results reveal that Ctf7p regulation is more complicated than previously envisioned and suggest that CDK acts in sister chromatid cohesion parallel to Ctf7p reactions.     

Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.     

Pch2 modulates chromatid partner choice during meiotic double-strand break repair in Saccharomyces cerevisiae

In most organisms, the segregation of chromosomes during the first meiotic division is dependent upon at least one crossover (CO) between each pair of homologous chromosomes. COs can result from chromosome double-strand breaks (DSBs) that are induced and preferentially repaired using the homologous chromosome as a template. The PCH2 gene of budding yeast is required to establish proper meiotic chromosome axis structure and to regulate meiotic interhomolog DSB repair outcomes. These roles appear conserved in the mouse ortholog of PCH2, Trip13, which is also involved in meiotic chromosome axis organization and the regulation of DSB repair. Using a combination of genetic and physical assays to monitor meiotic DSB repair, we present data consistent with pch2Δ mutants showing defects in suppressing intersister DSB repair. These defects appear most pronounced in dmc1Δ mutants, which are defective for interhomolog repair, and explain the previously reported observation that pch2Δ dmc1Δ cells can complete meiosis. Results from genetic epistasis analyses involving spo13Δ, rad54Δ, and mek1/MEK1 alleles and an intersister recombination reporter assay are also consistent with Pch2 acting to limit intersister repair. We propose a model in which Pch2 is required to promote full Mek1 activity and thereby promotes interhomolog repair.     

Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae

A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.     

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.     

Ctf18 is required for homologous recombination-mediated double-strand break repair

The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1–Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G₂/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination.     

Sister chromatid cohesion in mitosis.

Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle. Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation. Proteins that induce and regulate the separation of sister chromatids have also been recently identified. (This review is an updated version of one that was published in Current Opinion in Cell Biology 1998, 10:769-775.)     

Sister chromatid cohesion in mitosis

Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle. Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation. Proteins that induce and regulate the separation of sister chromatids have also been recently identified.     

Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae.

During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.     

Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.     

RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300bp and 60bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.     

Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae.

Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.     

Calpain 2 is required for sister chromatid cohesion

Calpains form a family of Ca²⁺-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2 depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion.     

A SUMO-like domain protein, Esc2, is required for genome integrity and sister chromatid cohesion in Saccharomyces cerevisiae

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Delta cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Delta rrm3Delta cells also have a severe growth defect and accumulate DNA damage in late S/G(2). In contrast, esc2Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Delta cells.     

DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.     

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.     

Phosphorylation of Ago2 is required for its role in DNA double-strand break repair

Repair of DNA double-strand break(DSB) is critical for the maintenance of genome integrity. A class of DSB-induced small RNAs(di RNAs) has been shown to play an important role in DSB repair. In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR). Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions. However, the role of Ago2 phosphorylation in DNA damage repair is unexplored. Here, we show that S672, S828, T830, and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR). S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency. We further show that defective association of Ago2 S672 A variant with DSB sites, instead of defects in di RNA and Rad51 binding, may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.     

The ACF1 complex is required for DNA double-strand break repair in human cells

DNA double-strand breaks (DSBs) are repaired via?nonhomologous end-joining (NHEJ) or homologous?recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.     

Sister chromatid cohesion and recombination in meiosis

Sister chromatids are associated from their formation until their disjunction. Cohesion between sister chromatids is provided by protein complexes, of which some components are conserved across the kingdoms and between the mitotic and meiotic cell cycles. Sister chromatid cohesion is intimately linked to other aspects of chromosome behaviour and metabolism, in particular chromosome condensation, recombination and segregation. Recombination, sister chromatid cohesion and the relation between the two processes must be regulated differently in mitosis and meiosis. In meiosis, cohesion and recombination are modified in such a way that reciprocal exchange and reductional segregation of homologous chromosomes are ensured. Received: 11 October 1999; in revised form: 3 December 1999 / Accepted: 6 December 1999