다중혈관 관상동맥 환자에서 y-문합을 이용하여 양쪽 내흉동맥만을 사용한 우회술의 조기 성적

▪ Abstract DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from a...

DNA topoisomerases: structure, function, and mechanism.

Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.

https://www.med.nyu.edu/klein/PDFs/annurev.biochem.77.061306.125255.pdf

Mechanism of eukaryotic homologous recombination.

EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

/pdf/automated-eukaryotic-gene-structure-annotation-using-3fo28wpira.pdf

Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.

https://science.sciencemag.org/content/sci/308/5721/551.full.pdf

ATM Activation by DNA Double-Strand Breaks Through the Mre11-Rad50-Nbs1 Complex

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dube (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present computational and experimental studies showing that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature to 29 °C led to a stabilization of two FLCN missense proteins, and for one variant (R362C), FLCN function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization of certain disease-linked variants may hold therapeutic potential.

/pdf/folliculin-variants-linked-to-birt-hogg-dube-syndrome-are-3fd59rfyku.pdf

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation

Genetic recombination relies on a number of biochemical activities that must be present at the right time and place in order for two DNA molecules to be recombined properly. Recent advances in real-time fluorescence microscopy provide us with a glimpse of homologous recombination taking place in living cells. These approaches reveal that homologous recombination is highly choreographed in vivo with its spatio-temporal organization being dependent on both cell cycle phase and the nature of the initiating DNA lesion. In this chapter, we review the cell biology of homologous recombination in mitotic cells with the main focus on the yeast Saccharomyces cerevisiae but also drawing parallels to other eukaryotic organisms.

The cell biology of mitotic recombination in Saccharomyces cerevisiae

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair the damaged recipient DNA, HR requires an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulating this transfer of information is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH) in diploid organisms, and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR but has a limited role in repairing DNA. Instead, several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate the flow of information between donor and recipient DNA molecules post DNA repair. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information after DNA repair. Our data indicates that RDH54 regulates DNA sequence exchange between chromosomes by limiting the disruption of Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 also protects Rad51 filaments, acting in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, displays a different distribution of information exchange outcomes than rdh54Δ cells. From these results, we propose a model for how Rdh54 may effectively regulate information transfer during homologous recombination.

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.

A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis

Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction be- tween the transcription elongation factor Bur1-Bur2 and repli- cation protein A (RPA), the eukaryotic single-stranded DNA- binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-C mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The tran- scription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.

Michael Lisby

Papers

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation

The cell biology of mitotic recombination in Saccharomyces cerevisiae

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis

The Transcription Elongation Factor Bur1-Bur2 Interacts with Replication Protein A and Maintains Genome Stability during