The RAD51 S181P mutation shortens lifespan of female mice.

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair.

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

ZNF212 promotes genomic integrity through direct interaction with TRAIP.

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.

PWWP2B promotes DNA end resection and homologous recombination.

Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.

NSMF promotes the replication stress-induced DNA damage response for genome maintenance.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.

ATAD5 suppresses centrosome over-duplication by regulating UAF1 and ID1.

Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.

RECQL5 and BLM exhibit divergent functions in cells defective for the Fanconi anemia pathway.

Fanconi anemia (FA) patients exhibit bone marrow failure, developmental defects and cancer. The FA pathway maintains chromosomal stability in concert with replication fork maintenance and DNA double strand break (DSB) repair pathways including RAD51-mediated homologous recombination (HR). RAD51 is a recombinase that maintains replication forks and repairs DSBs, but also rearranges chromosomes. Two RecQ helicases, RECQL5 and Bloom syndrome mutated (BLM) suppress HR through nonredundant mechanisms. Here we test the impact deletion of RECQL5 and BLM has on mouse embryonic stem (ES) cells deleted for FANCB, a member of the FA core complex. We show that RECQL5, but not BLM, conferred resistance to mitomycin C (MMC, an interstrand crosslinker) and camptothecin (CPT, a type 1 topoisomerase inhibitor) in FANCB-defective cells. RECQL5 suppressed, while BLM caused, breaks and radials in FANCB-deleted cells exposed to CPT or MMC, respectively. RECQL5 protected the nascent replication strand from MRE11-mediated degradation and restarted stressed replication forks in a manner additive to FANCB. By contrast BLM restarted, but did not protect, replication forks in a manner epistatic to FANCB. RECQL5 also lowered RAD51 levels in FANCB-deleted cells at stressed replication sites implicating a rearrangement avoidance mechanism. Thus, RECQL5 and BLM impact FANCB-defective cells differently in response to replication stress with relevance to chemotherapeutic regimes.

Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks.

BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3' overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2(lex1/lex2)) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.

Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks.

BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3' overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2(lex1/lex2)) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.

Two replication fork maintenance pathways fuse inverted repeats to rearrange chromosomes.

Replication fork maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation. Potential causal mechanisms are homologous recombination and error-free postreplication repair (EF-PRR). Homologous recombination repairs damage-induced DNA double-strand breaks (DSBs) and single-ended DSBs within replication. To facilitate homologous recombination, the recombinase RAD51 and mediator BRCA2 form a filament on the 3' DNA strand at a break to enable annealing to the complementary sister chromatid while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination. Homologous recombination also stabilizes and restarts replication forks without a DSB. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6-RAD18 and UBC13-MMS2-RAD5 ubiquitin ligase complexes. Some components are common to both homologous recombination and EF-PRR such as RAD51 and RAD18. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched, repeats. By contrast, ultraviolet light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3'→5' exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to ultraviolet light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling in response to nucleotide depletion. Interestingly, replication fork stalling induced fusion for identical and mismatched repeats, implicating faulty replication as a causal mechanism for both pathways.

Defining a genotoxic profile with mouse embryonic stem cells.

Many genotoxins are found in the environment from synthetic to natural, yet very few have been studied in depth. This means we fail to understand many molecules that damage DNA, we do not understand the type of damage they cause and the repair pathways required to correct their lesions. It is surprising so little is known about the vast majority of genotoxins since they have potential to cause disease from developmental defects to cancer to degenerative ailments. By contrast, some of these molecules have commercial and medical potential and some can be weaponized. Therefore, we need a systematic method to efficiently generate a genotoxic profile for these agents. A genotoxic profile would include the type of damage the genotoxin causes, the pathways used to repair the damage and the resultant mutations if repair fails. Mouse embryonic stem (ES) cells are well suited for identifying pathways and mutations. Mouse ES cells are genetically tractable and many DNA repair mutant cells are available. ES cells have a high mitotic index and form colonies so experiments can be completed quickly and easily. Furthermore, ES cells have robust DNA repair pathways to minimize genetic mutations at a particularly vulnerable time in life, early development when a mutation in a single cell could ultimately contribute to a large fraction of the individual. After an initial screen, other types of cells and mouse models can be used to complement the analysis. This review discusses the merging field of genotoxic screens in mouse ES cells that can be used to discover and study potential genotoxic activity for chemicals commonly found in our environment.

RAD51 mutants cause replication defects and chromosomal instability.

RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.

The phenotype of FancB-mutant mouse embryonic stem cells.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

High-throughput knock-in coupling gene targeting with the HPRT minigene and Cre-mediated recombination.

Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to phenotype; yet, for most SNPs their potential influence is unknown. Here, we present a technique in mouse embryonic stem cells that enables high-throughput knock-in (the placement of coding sequences adjacent to a specific endogenous promoter). Our methodology utilizes gene targeting with a combination of two selection cassettes (SAbetageo and the HPRT minigene) along with site-specific recombinases (Cre/loxP and FLP/FRT) to efficiently introduce multiple DNA sequences, including enhanced green fluorescent protein (eGFP), adjacent to the DNA topoisomerase 3beta (Top3beta) promoter. This technology enables rapid and efficient introduction of DNA sequences to a specific location and advances high-throughput analysis of many SNPs with control for expression and genetic background.

HPRT minigene generates chimeric transcripts as a by-product of gene targeting.

The HPRT minigene is a selection cassette used for gene targeting in mouse embryonic stem (ES) cells and, it is unique since selection may be applied for its presence and absence. This minigene has two exon clusters separated by a small intron and splicing sequences. We find these exon clusters splice into exons from the target gene forming two different classes of chimeric transcripts. The first class is expressed by the endogenous promoter and includes upstream target gene exons spliced into minigene exons 3-8. The second class is expressed by the minigene's PGK promoter and includes minigene exons 1-2 spliced into downstream target gene exons. These chimeric transcripts may produce chimeric proteins that could influence phenotype. Therefore, we have designed two floxed HPRT minigenes that permit removal of either the 5' half of the minigene or the entire minigene via Cre-mediated recombination.

Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination.

The breast cancer susceptibility protein, Brca2 and the RecQ helicase, Blm (Bloom syndrome mutated) are tumor suppressors that maintain genome integrity, at least in part, through homologous recombination (HR). Brca2 facilitates HR by interacting with Rad51 in multiple regions, the BRC motifs encoded by exon 11 and a single domain encoded by exon 27; however, the exact importance of these regions is not fully understood. Blm also interacts with Rad51 and appears to suppress HR in most circumstances; however, its yeast homologue Sgs1 facilitates HR in response to some genotoxins. To better understand the biological importance of these two proteins, we performed a genotoxic screen on mouse embryonic stem (ES) cells impaired for either Brca2 or Blm to establish their genotoxic profiles (a cellular dose-response to a wide range of agents). This is the first side-by-side comparison of these two proteins in an identical genetic background. We compared cells deleted for Brca2 exon 27 to cells reduced for Blm expression and find that the Brca2- and Blm-impaired cells exhibit genotoxic profiles that reflect opposing activities during HR. Cells deleted for Brca2 exon 27 are hypersensitive to gamma-radiation, streptonigrin, mitomycin C and camptothecin and mildly resistant to ICRF-193 which is similar to HR defective cells null for Rad54. By contrast, Blm-impaired cells are hypersensitive to ICRF-193, mildly resistant to camptothecin and mitomycin C and more strongly resistant to hydroxyurea. These divergent profiles support the notion that Brca2 and Blm perform opposing functions during HR in mouse ES cells.

Determination of genes related to gastrointestinal tract origin cancer cells using a cDNA microarray.

PURPOSE: We evaluated the genome-wide gene expression profiles of various cancer cell lines to identify the gastrointestinal tract cancer cell-related genes. EXPERIMENTAL DESIGN: Gene expression profilings of 27 cancer cell lines and 9 tissues using 7.5K human cDNA microarrays in indirect design with Yonsei reference RNA composed of 11 cancer cell line RNAs were done. The significant genes were selected using significant analysis of microarray in various sets of data. The selected genes were validated using real-time PCR analysis. RESULTS: After intensity-dependent, within-print-tip normalization by loess method, we observed that expression patterns of cell lines and tissues were substantially different, divided in two discrete clusters. Next, we selected 115 genes that discriminate gastrointestinal cancer cell lines from others using significant analysis of microarray. Among the expression profiles of five gastric cancer cell lines, 66 genes were identified as differentially expressed genes related to metastatic phenotype. YCC-16, which was established from the peripheral blood of one advanced gastric cancer patient, produced a unique gene expression pattern resembling the profiles of lymphoid cell lines. Quantitative real-time reverse transcription-PCR results of selected genes, including PXN, KRT8, and ITGB5, were correlated to microarray data and successfully discriminate the gastrointestinal tract cancer cell lines from hematologic malignant cell lines. CONCLUSIONS: A gene expression database could serve as a useful source for the further investigation of cancer biology using the cell lines.

Molecular cloning and characterization of a novel inhibitor of apoptosis protein from Xenopus laevis.

A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRalpha through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.