Sister Chromatid Homologous Recombination Research Articles

A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

Errors during DNA replication are one likely cause of gross chromosomal rearrangements (GCRs). Here, we analyze the role of RNase H2, which functions to process Okazaki fragments, degrade transcription intermediates, and repair misincorporated ribonucleotides, in preventing genome instability. The results demonstrate that rnh203 mutations result in a weak mutator phenotype and cause growth defects and synergistic increases in GCR rates when combined with mutations affecting other DNA metabolism pathways, including homologous recombination (HR), sister chromatid HR, resolution of branched HR intermediates, postreplication repair, sumoylation in response to DNA damage, and chromatin assembly. In some cases, a mutation in RAD51 or TOP1 suppressed the increased GCR rates and/or the growth defects of rnh203Δ double mutants. This analysis suggests that cells with RNase H2 defects have increased levels of DNA damage and depend on other pathways of DNA metabolism to overcome the deleterious effects of this DNA damage.

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl

DNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister chromatid homologous recombination (SCR). Here we show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent small ubiquitin-like modifier (SUMO) accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the nonsumoylatable Scc1 mutant (15KR) maintain sister chromatid cohesion during mitosis but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation.

Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl

DNA double‐strand break (DSB) is the most deleterious form of DNA damage and can cause chromosome translocations that drive cancer formation. Homologous recombination (HR) is a key DSB repair pathway. To maintain heterozygosity of the genome, sister‐chromatid homologous recombination (SCR) is preferred to HR between two homologous chromosomes. In human cells, two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, facilitate SCR. We have previously shown that the Smc5/6 subunit Mms21 functions as a SUMO ligase. In this study, we demonstrate that Mms21 sumoylates the cohesin subunit Scc1 at multiple sites. SUMO2/3 is accumulated on laser‐induced DNA damages, and this accumulation is dependent on Smc5/6 and cohesin during the S/G2 phase when DSBs are primarily repaired through SCR. Human cells expressing a non‐sumoylatable Scc1 mutant (15KR) maintain proper sister‐chromatid cohesion during mitosis, but are defective in SCR. These results show that Scc1 sumoylation by Mms21 is essential for SCR. On the other hand, Scc1 15KR is still recruited to laser‐induced DNA damage sites. Depletion of Wapl, the negative cohesin regulator, rescues SCR defects and IR sensitivity of Mms21‐deficient or Scc1 15KR‐expressing cells. Therefore, our results suggest that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after the initial cohesin loading at DSBs in human cells.

The Smc5/6 complex and the difficulties cutting the ties of twin sisters

The Smc5/6 complex and the difficulties cutting the ties of twin sisters

Abstract 3158: Evidence for a role of EWS in alternative splicing in Ewing's sarcoma

Abstract Ewing's sarcoma family tumors (ESFT) are characterized in 85% of cases by the oncogenic fusion protein EWS-FLI1 resulting from a chromosomal translocation. While FLI1 is an ETS transcription factor, EWS belongs to the evolutionarily conserved FET family of RNA-binding proteins. So far, functional studies of oncogenic EWS fusion proteins have concentrated on their presumed role as transcriptional regulators. However, chimeric FET proteins are usually co-expressed with their intact counterparts and previous protein interaction studies have shown that EWS and EWS-FLI1 interact with each other. Therefore, it is possible that functional interference with normal EWS function accounts for part of the oncogenic activities of EWS-FLI1. However, the role of EWS in post-transcriptional and transcriptional gene regulation is poorly defined. Since FET proteins associate with a number of RNA processing factors, we investigated the influence of modulated EWS expression on genome wide splicing using Affymetrix HuEx-1.0stv2 arrays. For that purpose, we used two model systems: a unique ESFT cell line lacking endogenous EWS was studied upon stable restoration of EWS expression, and an ESFT cell line allowing for inducible RNAi-mediated EWS-FLI1 suppression was analyzed upon transient EWS knockdown. As a result, we found significant differences between cells with and without EWS expression in splicing patterns as well as alternative promoter usage for more than 50 genes. GO analysis of the top ranking genes is consistent with previously suggested functions of the FET protein family such as DNA repair, chromatin structure and RNA transcription. The alternative splicing patterns were verified by RT-qPCR of candidate genes which are currently tested for their functional relevance to ESFT. One of these candidate genes is structural maintainance of chromosome 5 (SMC5). As a core component of the SMC5-SMC6 complex it is involved in sister chromatid homologous recombination by recruiting the SMC1-SMC3 cohesin complex to DNA double-strand breaks during anaphase. Ongoing studies concentrate on the influence of EWS-FLI1 on EWS-dependent alternative RNA processing patterns in these model systems. This study is supported by grant P20665-B12 of the Austrian Science Fund FWF Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3158.

Sumoylation and the Structural Maintenance of Chromosomes (Smc) 5/6 Complex Slow Senescence through Recombination Intermediate Resolution

Telomeres are repetitive nucleoprotein structures that cap the ends of chromosomes. Without telomerase, telomeres shorten with replication and eventually signal cell cycle arrest (cell senescence). Homologous recombination (HR)-based mechanisms slow senescence, and distinct HR mechanisms support the growth of the rare survivors of senescence. Here, we report novel roles for the post-translational modification of small ubiquitin-like modifier (SUMO) in regulating the rate of senescence in Saccharomyces cerevisiae telomerase mutants. We identify Mms21 as the relevant SUMO E3 ligase and demonstrate that cells lacking Mms21-dependent sumoylation accumulate HR intermediates selectively at telomeres during senescence. One target of Mms21-dependent sumoylation is the cohesin- and condensin-related Smc5-Smc6 complex (Smc5/6). We show that hypomorphic smc5 or smc6 alleles exhibit phenotypes similar to mms21 sumoylation-deficient mutants with regard to senescence and the accumulation of unresolved HR intermediates. Further, we provide evidence that Mms21 and Smc5/6 prevent aberrant recombination between sister telomeres and also globally facilitate resolution of sister chromatid HR intermediates.

Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks.

The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex is thought to promote HR by maintaining the close proximity of sister chromatids at DSBs. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this is unclear. Here, we show that RNAi-mediated knockdown of the SMC5/6 complex components in human cells increases the efficiency of gene targeting due to a specific requirement for hSMC5/6 in sister chromatid HR. Knockdown of the hSMC5/6 complex decreases sister chromatid HR, but does not reduce nonhomologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. The hSMC5/6 complex is itself recruited to nuclease-induced DSBs and is required for the recruitment of cohesin to DSBs. Our results establish a mechanism by which the hSMC5/6 complex promotes DNA repair and suggest a novel strategy to improve the efficiency of gene targeting in mammalian somatic cells.