Kirill S. Lobachev

The Mre11 Complex Is Required for Repair of Hairpin-Capped Double-Strand Breaks and Prevention of Chromosome Rearrangements

Inverted repeats (IRs) that can form a hairpin or cruciform structure are common in the human genome and may be sources of instability. An IR involving the human Alu sequence (Alu-IR) has been studied as a model of such structures in yeast. We found that an Alu-IR is a mitotic recombination hotspot requiring MRE11/RAD50/XRS2 and SAE2. Using a newly developed approach for mapping rare double-strand breaks (DSBs), we established that induction of recombination results from breaks that are terminated by hairpins. Failure of the mre11, rad50, xrs2, and sae2 mutants to process the hairpins blocks recombinational repair of the DSBs and leads to generation of chromosome inverted duplications. Our results suggest an additional role for the Mre11 complex in maintaining genome stability.

Genes required for ionizing radiation resistance in yeast

The ability of Saccharomyces cerevisiae to tolerate ionizing radiation damage requires many DNA-repair and checkpoint genes, most having human orthologs. A genome-wide screen of diploid mutants homozygous with respect to deletions of 3,670 nonessential genes revealed 107 new loci that influence γ-ray sensitivity. Many affect replication, recombination and checkpoint functions. Nearly 90% were sensitive to other agents, and most new genes could be assigned to the following functional groups: chromatin remodeling, chromosome segregation, nuclear pore formation, transcription, Golgi/vacuolar activities, ubiquitin-mediated protein degradation, cytokinesis, mitochondrial activity and cell wall maintenance. Over 50% share homology with human genes, including 17 implicated in cancer, indicating that a large set of newly identified human genes may have related roles in the toleration of radiation damage.

Chromosomal Translocations in Yeast Induced by Low Levels of DNA Polymerase: A Model for Chromosome Fragile Sites

In the yeast Saccharomyces cerevisiae, reduced levels of the replicative alpha DNA polymerase result in greatly elevated frequencies of chromosome translocations and chromosome loss. We selected translocations in a small region of chromosome III and found that they involve homologous recombination events between yeast retrotransposons (Ty elements) on chromosome III and retrotransposons located on other chromosomes. One of the two preferred sites of these translocations on chromosome III involve two Ty elements arrayed head-to-head; disruption of this site substantially reduces the rate of translocations. We demonstrate that this pair of Ty elements constitutes a preferred site for double-strand DNA breaks when DNA replication is compromised, analogous to the fragile sites observed in mammalian chromosomes.

Migrating bubble during break-induced replication drives conservative DNA synthesis

The repair of chromosomal double strand breaks (DSBs) is crucial for the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break-induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobases of DNA from a donor molecule all the way through its telomere. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis. BIR uses most known replication proteins to copy large portions of DNA, similar to S-phase replication. It has therefore been suggested that BIR proceeds by semiconservative replication; however, the model of a bona fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1,000-fold increase in mutation rate compared to normal replication. Here we demonstrate that in budding yeast the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the marked increase in BIR-associated mutations. We propose that the BIR mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes, including humans.

Replication stalling at unstable inverted repeats: Interplay between DNA hairpins and fork stabilizing proteins

DNA inverted repeats (IRs) are hotspots of genomic instability in both prokaryotes and eukaryotes. This feature is commonly attributed to their ability to fold into hairpin- or cruciform-like DNA structures interfering with DNA replication and other genetic processes. However, direct evidence that IRs are replication stall sites in vivo is currently lacking. Here, we show by 2D electrophoretic analysis of replication intermediates that replication forks stall at IRs in bacteria, yeast, and mammalian cells. We found that DNA hairpins, rather than DNA cruciforms, are responsible for the replication stalling by comparing the effects of specifically designed imperfect IRs with varying lengths of their central spacer. Finally, we report that yeast fork-stabilizing proteins, Tof1 and Mrc1, are required to counteract repeat-mediated replication stalling. We show that the function of the Tof1 protein at DNA structure-mediated stall sites is different from its previously described effect on protein-mediated replication fork barriers.

The Pattern of Gene Amplification Is Determined by the Chromosomal Location of Hairpin-Capped Breaks

DNA palindromes often colocalize in cancer cells with chromosomal regions that are predisposed to gene amplification. The molecular mechanisms by which palindromes can cause gene amplification are largely unknown. Using yeast as a model system, we found that hairpin-capped double-strand breaks (DSBs) occurring at the location of human Alu-quasipalindromes lead to the formation of intrachromosomal amplicons with large inverted repeats (equivalent to homogeneously staining regions in mammalian chromosomes) or extrachromosomal palindromic molecules (equivalent to double minutes [DM] in mammalian cells). We demonstrate that the specific outcomes of gene amplification depend on the applied selection, the nature of the break, and the chromosomal location of the amplified gene relative to the site of the hairpin-capped DSB. The rules for the palindrome-dependent pathway of gene amplification defined in yeast may operate during the formation of amplicons in human tumors.

Inverted Alu repeats unstable in yeast are excluded from the human genome

The nearly one million Alu repeats in human chromosomes are a potential threat to genome integrity. Alus form dense clusters where they frequently appear as inverted repeats, a sequence motif known to cause DNA rearrangements in model organisms. Using a yeast recombination system, we found that inverted Alu pairs can be strong initiators of genetic instability. The highly recombinagenic potential of inverted Alu pairs was dependent on the distance between the repeats and the level of sequence divergence. Even inverted Alus that were 86% homologous could efficiently stimulate recombination when separated by <20 bp. This stimulation was independent of mismatch repair. Mutations in the DNA metabolic genes RAD27 (FEN1), POL3 (polymerase δ) and MMS19 destabilized widely separated and diverged inverted Alus. Having defined factors affecting inverted Alu repeat stability in yeast, we analyzed the distribution of Alu pairs in the human genome. Closely spaced, highly homologous inverted Alus are rare, suggesting that they are unstable in humans. Alu pairs were identified that are potential sites of genetic change.

Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex.

Chromosome aberrations are common outcomes of exposure to DNA-damaging agents or altered replication events and are associated with various diseases and a variety of carcinomas, including leukemias, lymphomas, sarcomas, and epithelial tumors. The incidence of aberrations can be greatly increased as a result of defects in DNA repair pathways. Although there is considerable information about the molecular events associated with the induction and repair of a double-strand break (DSB), little is known about the events that ultimately lead to translocations or deletions through the formation of chromosome breaks or the dissociation of broken ends. We describe a system for visualizing DNA ends at the site of a DSB in living cells. After induction of the break, DNA ends flanking the DSB site in wild-type cells remained adjacent. Loss of a functional RMX complex (Rad50/Mre11/Xrs2) or a mutation in the Rad50 Zn-hook structure resulted in DNA ends being dispersed in approximately 10%-20% of cells. Thus, the RMX complex holds broken ends together and counteracts mitotic spindle forces that can be destructive to damaged chromosomes.

Large-Scale Expansions of Friedreich's Ataxia GAA Repeats in Yeast

Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats responsible for the human disease Friedreichs ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeats orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk

ABSTRACT Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3′→5′ exonuclease domain of DNA polymerase δ. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined.