Induction Of Genomic Instability Research Articles

Impact of UV Radiation on Genome Stability and Human Health.

Gradual depletion of the atmospheric ozone layer during the past few years has increased the incidence of solar UV radiation specifically the UV-C on earth's surface is one of the major environmental concerns because of the harmful effects of this radiation in all forms of life. The solar UV radiation including the harmful wavelength range of UV-B (280-320nm) represents a significant climatic stress for both animals and plants, causing damage to the fundamental biomolecules such as DNA, proteins and lipids, thus activating genotoxic stress and induces genome instability. When DNA absorbs UV-B light, energy from the photon causes covalent linkages to form between adjacent pyrimidine bases, creating photoproducts, primarily cyclobutane pyrimidine dimers (CPDs) and pyrimidine-6,4-pyrimidinone photoproduct (6,4PPs). Pyrimidine dimers create distortions in the DNA strands and therefore can inhibit DNA replication as well transcription. Lack of efficient repair of UV-induced DNA damage may induce the formation of DNA double stand breaks (DSBs), one of the serious forms of damage in DNA double helix, as well as oxidative damage. Unrepaired DSBs in the actively dividing somatic cells severely affect cell growth and development, finally results in loss of cell viability and development of various diseases, such as cancer in man.This chapter mainly highlights the incidence of solar UV-radiation on earth's surface along with the formation of major types of UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and finally our present understanding on the impact on solar UV radiation on human health.

Bacterial Infection and Associated Cancers.

Bacterial infections were traditionally not considered as major causes of cancer. However, increasing evidence in the past decades has suggested that several cancers are highly associated with bacterial infection. The bacterial infections have evolved some unique strategies including lateral gene transfer, biofilm and microbiome to induce genome instability and chronic inflammation, as well as escape of immune surveillance for carcinogenesis. Here we summarize and highlight the recent progress on understanding of how bacterial infection plays a role in tumor formation and malignancy.

Genotoxic potential of the binary mixture of cyanotoxins microcystin-LR and cylindrospermopsin

Increased eutrophication of water bodies promotes cyanobacterial blooming that is hazardous due to the production of various bioactive compounds. Microcystin-LR (MCLR) is among the most widespread cyanotoxins classified as possible human carcinogen, while cylindrospermopsin (CYN) has only recently been recognized as health concern. Both cyanotoxins are genotoxic; however, the mechanisms of their action differ. They are ubiquitously present in water environment and are often detected together. Therefore, we studied genotoxic potential of the binary mixture of these cyanotoxins. Human hepatoma cells (HepG2) were exposed to a single dose of MCLR (1 μg/mL), graded doses of CYN (0.01-0.5 μg/mL), and their combinations. Comet and Cytokinesis block micronucleus assays were used to detect induction of DNA strand breaks (sb) and genomic instability, respectively, along with the transcriptional analyses of the expression of selected genes involved in xenobiotic metabolism, immediate/early cell response and DNA-damage response. MCLR induced DNA sb that were only transiently present after 4 h exposure, whereas CYN, after 24 h exposure, induced DNA sb and genomic instability. The MCLR/CYN mixture induced DNA sb after 24 h exposure, but to lesser extent as CYN alone. On the other hand, induction of genomic instability by the MCLR/CYN mixture was comparable to that induced by CYN alone. In addition, patterns of changes in the expression of selected genes induced by the MCLR/CYN mixture were not significantly different from those induced by CYN alone. Our results indicate that CYN exerts higher genotoxic potential than MCLR and that genotoxic potential of the MCLR/CYN mixture is comparable to that of CYN alone.

Abstract 3580: Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements

Abstract Topoisomerase 2 (Top2) is the target of active anti-cancer agents such as etoposide and doxorubicin. These drugs interfere with the Top2 catalytic cycle and lead to trapping of the enzyme as a covalent protein: DNA complex. This trapped covalent complex is a unique DNA lesion that includes DNA strand breaks and covalent protein adducts at the site of the breaks. While Top2 mediated DNA damage is the major mechanism for tumor cell killing, it is also responsible for drug-induced translocations that can lead to secondary malignancies. We have developed several novel systems for assessing Top2-mediated genome instability using yeast as a model system. We developed repair proficient yeast strains that accumulate high levels of etoposide and other Top2 targeting drugs. We selected for loss-of-function mutations in the yeast CAN1 gene that arise following etoposide treatment, and examined the induced mutations by DNA sequencing. We identified a unique spectrum of mutations generated by etoposide in yeast. One novel class of mutational events induced by etoposide is relatively large deletions (>300 bp) and tandem duplications. Most of these etoposide-induced events are flanked by 4-8 nucleotide direct repeats. To further characterize the mechanism of Top2 induced genome instability, we took advantage of a novel Top2 allele isolated in our laboratory. This Top2 allele, termed top2AR, exhibits elevated levels of drug independent DNA cleavage in vitro, and inviability when combined with yeast mutants defective in DNA double strand break repair. We found a similar spectrum of large deletions and tandem duplications flanked by 4-8 nucleotide direct repeats. Interestingly, we also found that the recovery of large deletions and insertions was enhanced in yeast cells lacking Tdp1, a gene that participates in the repair of protein:DNA covalent complexes. Our results illustrate a novel type of genome rearrangement that is mediated by covalent Top2 DNA damage. It is noteworthy that the deletions and insertions flanked by short direct repeats have not been previously observed with other classes of DNA damaging agents. We suggest that the unique properties of these lesions may also contribute to oncogenic translocations induced by etoposide and other Top2 targeting drugs. We are currently using next-generation sequencing of etoposide treated yeast cells and cells carrying the top2AR allele to obtain a more complete picture of the types of alterations induced by Top2 mediated DNA damage. Citation Format: Matthew Gilbertson, Radhika Patel, Karin C. Nitiss, John L. Nitiss. Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3580.

Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

Genome Stability Requires p53.

It is now clear that functional p53 is critical to protect the genome from alterations that lead to tumorigenesis. However, with the myriad of cellular stresses and pathways linked to p53 activation, much remains unknown about how p53 maintains genome stability and the proteins involved. The current understanding of the multiple ways p53 contributes to genome stability and how two of its negative regulators, Mdm2 and Mdmx, induce genome instability will be described.

Carcinogenesis associated with parasites other than Schistosoma, Opisthorchis and Clonorchis: A systematic review.

Only three helminths (Schistosoma haematobium, Opisthorchis viverrini and Clonorchis sinensis) are directly associated with carcinogenesis in humans whereas the role of other parasites in cancer remains unclear. This study aimed to perform a systematic review to identify recent insights in the role of other parasite infections in carcinogenesis. We conducted systematic searches of MEDLINE and EMBASE on July 2015. Our primary outcome was the association between parasitic infections and carcinogenesis. Out of 1,266 studies, 19 were selected for detailed evaluation (eight for helminths and 11 for protozoa). The mechanisms of helminth-induced cancer included chronic inflammation, sustained proliferation, modulation of the host immune system, reprogramming of glucose metabolism and redox signaling, induction of genomic instability and destabilization of suppressor tumor proteins, stimulation of angiogenesis, resisting cell death, and activation of invasion and metastasis. In addition to the current knowledge, the following parasites were found in cancers or tumors: Echinococcus, Strongyloides, Fasciola, Heterakis, Platynosomum and Trichuris. Additional parasites were found in this systematic review that could potentially be associated with cancers or tumors but further evidence is needed to elaborate a cause-effect relationship.

Telomere length assessment in leukocytes presents potential diagnostic value in patients with breast cancer.

Telomere shortening is associated with cancer development, primarily through the induction of genomic instability. The majority of studies have indicated that individuals with shorter blood telomeres may be at a higher risk of developing various types of cancer. There is increasing evidence that the study of the alterations in telomere length may improve cancer prognosis. The aim of the present study was to verify the use of telomere length parameters in the diagnostics of breast cancer stage. Telomere length was analyzed in the blood leukocytes of 52 patients with breast cancer relative to 47 control subjects using quantitative polymerase chain reaction. The effects of stage, grade, estrogen receptor, progesterone receptor and human epidermal growth factor 2 (HER2) status were assessed. The current study demonstrated that the average telomeric sequence length was significantly shorter in leukocytes from individuals diagnosed with a more severe stage of breast cancer (T2N1M0) than in leukocytes in the early stages of the disease (T1N0M0) (P=0.0207). Furthermore, the data indicated that telomeres in leukocytes derived from patients with HER2+ breast cancer were significantly longer compared with those with the HER2- type (P=0.0347). These results suggest that the assessment of telomeres in blood leukocytes may, at least partially, correspond with breast cancer staging and HER2 receptor status.

Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages

Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

Late-occurring chromosome aberrations and global DNA methylation in hematopoietic stem/progenitor cells of CBA/CaJ mice exposed to silicon (28Si) ions

Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to 28Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1cGy/min) to various doses of 300MeV/n 28Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p<0.01), indicating the induction of genomic instability after exposure of mice to 300MeV/n 28Si ions. Slight increases in the levels of 5mC were observed in all treatment groups, as compared to the sham-control level. In contrast, there was a significant reduction in levels of 5hmC (ANOVA, p<0.01). Since these endpoints were evaluated in the same mouse, our data suggested for the first time a link between a reduction in 5hmC and genomic instability in HSPC-derived myeloid colonies of CBA/CaJ mice exposed to 300MeV/n 28Si ions.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Developmental regulation of the Tetrahymena thermophila origin recognition complex.

The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.

Utility of microRNAs and siRNAs in cervical carcinogenesis.

MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

DNA damage signaling guards against perturbation of cyclin D1 expression triggered by low-dose long-term fractionated radiation.

Cyclin D1 expression is precisely controlled during cell-cycle progression. However, repeated exposure to low-dose fractionated radiation (FR) abrogates cell cycle-dependent cyclin D1 degradation by constitutive activation of AKT survival signaling in normal human fibroblasts. The resulting abnormal nuclear cyclin D1 accumulation induces defects in DNA replication and resulting DNA double-strand breaks, and is associated with induction of genomic instability in low-dose irradiated cells. Here, we investigated the role of DNA damage signaling against such perturbed cell-cycle control of cyclin D1 expression. Nuclear cyclin D1 accumulation was induced within 7 days after low-dose FR (0.01 Gy or 0.05 Gy per fraction) in ATM-deficient cells (AT5BIVA), but appeared later in AT5BIVA cells harboring human ATM cDNA. Thus, ATM prevents abnormal nuclear cyclin D1 accumulation at early time points after low-dose FR. We further demonstrated that ATM-mediated downregulation of protein phosphatase 2A activity caused activation of the AKT/cyclin D1 pathway after long-term FR. Perturbation of cyclin D1 expression induced Rad51 foci that indicate homologous recombination repair (HRR) in control cells, while ATM- and NBS1-deficient cells (GM7166) failed to induce Rad51 foci after long-term low-dose FR. After 21 days of FR, NBS1- and ATM-deficient cells showed a decrease in nuclear cyclin D1-positive cells, and an increase in apoptotic cells. Similarly, inhibition of ATM with KU55933 abrogated nuclear cyclin D1 accumulation by induction of apoptosis in ATM-complemented cells exposed to low-dose FR. In conclusion, we here demonstrate that ATM is involved in controlling cyclin D1 levels after low-dose FR. DNA damage signaling mitigates the harmful effects of low-dose long-term FR by suppression of cell death induced by perturbation of cyclin D1 expression.

Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic instability

SSX cancer/testis antigens are frequently expressed in melanoma tumors and represent attractive targets for immunotherapy, but their role in melanoma tumorigenesis has remained elusive. Here, we investigated the cellular effects of SSX2 expression. In A375 melanoma cells, SSX2 expression resulted in an increased DNA content and enlargement of cell nuclei, suggestive of replication aberrations. The cells further displayed signs of DNA damage and genomic instability, associated with p53-mediated G1 cell cycle arrest and a late apoptotic response. These results suggest a model wherein SSX2-mediated replication stress translates into mitotic defects and genomic instability. Arrest of cell growth and induction of DNA double-strand breaks was also observed in MCF7 breast cancer cells in response to SSX2 expression. Additionally, MCF7 cells with ectopic SSX2 expression demonstrated typical signs of senescence (i.e. an irregular and enlarged cell shape, enhanced β-galactosidase activity and DNA double-strand breaks). Since replication defects, DNA damage and senescence are interconnected and well-documented effects of oncogene expression, we tested the oncogenic potential of SSX2. Importantly, knockdown of SSX2 expression in melanoma cell lines demonstrated that SSX2 supports the growth of melanoma cells. Our results reveal two important phenotypes of ectopic SSX2 expression that may drive/support tumorigenesis: First, immediate induction of genomic instability, and second, long-term support of tumor cell growth.

Abstract LB-323: Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase

Abstract Colorectal cancer develops in the context of probably the most complex microenvironment in the human body: the colon microbiota comprised of &amp;gt;10¹³ bacteria, most of which are uncharacterized. Recently there has been great interest in defining the normal colon microbiome and how it is remodeled during disease states. Both commensal and infective enteropathogenic bacteria utilize a vast array of mechanisms to reduce inflammation in the gut, many of which are directed at the innate immune system, though undoubtedly, other cellular homeostasis systems are targeted. To limit inflammation bacterially encoded virulence proteins are injected into colon epithelial cells: many function by disrupting NFκB signaling. We have characterized the NLEE virulence protein which is encoded by EPEC bacteria and following injection by the T3SS, targets the TAB2 scaffold protein resulting in inactivation of the TAK1 kinase in the NFκB pathway. The Shao laboratory discovered that NLEE is an S-adenosyl-methionine (SAM)-dependent cysteine methyltransferase that methylates a single cysteine in the C4 zinc finger of TAB2 thereby unfolding and abolishing its function. The discovery of this first-in-class, Zinc Finger (ZF)-directed cysteine methytransferase, establishes the paradigm that eukaryotic ZFs can be subject to PTMs at the chelation residues that abolish finger function. We hypothesize that cysteine methylation of ZF domains is widespread and as yet, under appreciated PTM which regulates cellular signal transduction. The crystal structure of NLEE was solved and shows a novel SAM binding motif positioned in a deep cleft which confers a very high specificity for ZF binding and methylation. We searched for novel NLEE ZF-containing substrates in EPEC infected colon cells and discovered that the human DNA repair protein ZRANB3, a helicase which participates in the translesion synthesis repair pathway directly at replication forks, is a robust substrate for NLEE. Methylation of the C4 ZF of ZRANB3 is predicted to abolish its binding to K63-linked ubiquitinated-PCNA at stalled replication forks leading to fork collapse and replication stress. Additional NLEE substrates discovered in this DNA repair pathway include a PCNA-directed OTU class deubiquitinase and the NPL4 segregase complex, both of which limit repair functions. Together with recent work showing that bacterial virulence proteins can also down regulate the DNA mismatch repair system, these results suggest the remarkable conclusion that infectious colon bacteria have the capacity to abolish specific DNA repair systems in the colon epithelium. This work provides a unique and unexpected mechanistic link between the colon microbiome and genome stability in the colon. Whether a remodeled colon cancer specific microbiome contributes to tumor progression via induction of genome instability by inactivating DNA repair pathways is interesting to contemplate. Citation Format: Jayashree Karar, Hongzhuang Peng, Li Zhang, Qing Yao, Ilan Rosenshine, Feng Shao, Frank J. Rauscher. Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2014-LB-323

Replication initiation and genome instability: a crossroads for DNA and RNA synthesis.

Nuclear DNA replication requires the concerted action of hundreds of proteins to efficiently unwind and duplicate the entire genome while also retaining epigenetic regulatory information. Initiation of DNA replication is tightly regulated, rapidly firing thousands of origins once the conditions to promote rapid and faithful replication are in place, and defects in replication initiation lead to proliferation defects, genome instability, and a range of developmental abnormalities. Interestingly, DNA replication in metazoans initiates in actively transcribed DNA, meaning that replication initiation occurs in DNA that is co-occupied with tens of thousands of poised and active RNA polymerase complexes. Active transcription can induce genome instability, particularly during DNA replication, as RNA polymerases can induce torsional stress, formation of secondary structures, and act as a physical barrier to other enzymes involved in DNA metabolism. Here we discuss the challenges facing mammalian DNA replication, their impact on genome instability, and the development of cancer.

The role of the chlamydial effector CPAF in the induction of genomic instability

The role of CPAF in chlamydial virulence is justifiably a tangle of confusion. CPAF is a potent and promiscuous cysteine protease capable of cleaving many proteins (Zhong, 2011). Over the 13 years since the original characterization of CPAF in the laboratory of Guaming Zohng (Zhong et al ., 2001) chlamydial researchers have contributed a long list of identified CPAF cleavable target proteins of both chlamydial and host origin (A Conrad et al ., 2013). However, careful studies by the Tan and Sutterlin group convincingly demonstrated that the majority of CPAF activity in infected cells is actually sequestered in the chlamydial inclusion and that biochemical assays relying on cell homogenization in many cases released the sequestered CPAF allowing degradation of ‘targets’ that would not interact with CPAF under biological conditions (Chen et al ., 2012). Thus, the question becomes not what host proteins does CPAF cleave but what host proteins does CPAF have access to and when does it gain this access. New data from the investigation of growth and virulence phenotypes of two mutant strains of chlamydia null for CPAF activity isolated by Emily Snavely in the Valdivia laboratory have added critical observations that have evolved our understanding of the role of CPAF in pathogenesis (Snavely et al ., 2014). The Valdivia laboratory observed that CPAF null strains follow the same developmental cycle and produce normal appearing …

Canonical non-homologous end joining in mitosis induces genome instability and is suppressed by M-phase-specific phosphorylation of XRCC4.

DNA double-strand breaks (DSBs) can be repaired by one of two major pathways—non-homologous end-joining (NHEJ) and homologous recombination (HR)—depending on whether cells are in G1 or S/G2 phase, respectively. However, the mechanisms of DSB repair during M phase remain largely unclear. In this study, we demonstrate that transient treatment of M-phase cells with the chemotherapeutic topoisomerase inhibitor etoposide induced DSBs that were often associated with anaphase bridge formation and genome instability such as dicentric chromosomes. Although most of the DSBs were carried over into the next G1 phase, some were repaired during M phase. Both NHEJ and HR, in particular NHEJ, promoted anaphase-bridge formation, suggesting that these repair pathways can induce genome instability during M phase. On the other hand, C-terminal-binding protein interacting protein (CtIP) suppressed anaphase bridge formation, implying that CtIP function prevents genome instability during mitosis. We also observed M-phase-specific phosphorylation of XRCC4, a regulatory subunit of the ligase IV complex specialized for NHEJ. This phosphorylation required cyclin-dependent kinase (CDK) activity as well as polo-like kinase 1 (Plk1). A phosphorylation-defective XRCC4 mutant showed more efficient M-phase DSB repair accompanied with an increase in anaphase bridge formation. These results suggest that phosphorylation of XRCC4 suppresses DSB repair by modulating ligase IV function to prevent genome instability during M phase. Taken together, our results indicate that XRCC4 is required not only for the promotion of NHEJ during interphase but also for its M-phase-specific suppression of DSB repair.