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Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health.

The accurate repair of DNA damage specifically the chromosomal double-strand breaks (DSBs) arising from exposure to physical or chemical agents, such as ionizing radiation (IR) and radiomimetic drugs is critical in maintaining genomic integrity. The DNA DSB response and repair is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes specifically histone modifications which impact cell-cycle checkpoints through enzymatic activities to repair the broken DNA ends. Various histone posttranslational modifications such as phosphorylation, acetylation, methylation and ubiquitylation have been shown to play a role in DNA damage repair. Recent studies have provided important insights into the role of histone-specific modifications in sensing DNA damage and facilitating the DNA repair. Histone modifications have been shown to determine the pathway choice for repair of DNA DSBs. This review will summarize the role of important histone acetyltransferases MOF and Tip60 mediated acetylation in repair of DNA DSBs in eukaryotic cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c- fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ -H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery., Competing Interests: Competing interestsThe authors declare no competing interests.

MOF (males absent on the first) was initially identified as a dosage compensation factor in Drosophila that acetylates lysine 16 of histone H4 (H4K16ac) and increased gene transcription from the single copy male X-chromosome. In humans, however, the ortholog of Drosophila MOF has been shown to interact with a range of proteins that extend its potential significance well beyond transcription. For example, recent results indicate MOF is an upstream regulator of the ATM (ataxia-telangiectasia mutated) protein, the loss of which is responsible for ataxia telangiectasia (AT). ATM is a key regulatory kinase that interacts with and phosphorylates multiple substrates that influence critical, cell-cycle control and DNA damage repair pathways in addition to other pathways. Thus, directly or indirectly, MOF may be involved in a wide range of cellular functions. This review will focus on the contribution of MOF to cellular DNA repair and new results that are beginning to examine the in vivo physiological role of MOF., Competing Interests: COI: Authors declare that they have no conflict of interest., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

β2-Spectrin (β2SP/SPTBN1, gene SPTBN1) is a key TGF-β/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-β signaling and can contribute to liver cancer development. Here we report that cells deficient in β2-Spectrin (β2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), β2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, β2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in β2SP deficient cells. We propose that β2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination., Competing Interests: No potential conflicts of interest were disclosed.

Several classes of inhibitors of the mammalian target of rapamycin (mTOR) have been developed based on its central role in sensing growth factor and nutrient levels to regulate cellular metabolism. However, its ATP-binding site closely resembles other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, resulting in reactivity with these targets that may also be therapeutically useful. The ATP-competitive mTOR inhibitor, Torin2, shows biochemical activity against the DNA repair-associated proteins ATM, ATR and DNA-PK, which raises the possibility that Torin2 and related compounds might radiosensitize cancerous tumors. In this study Torin2 was also found to enhance ionizing radiation-induced cell killing in conditions where ATM was dispensable, confirming the requirement for multiple PIKK targets. Moreover, Torin2 did not influence the initial appearance of γ-H2AX foci after irradiation but significantly delayed the disappearance of radiation-induced γ-H2AX foci, indicating a DNA repair defect. Torin2 increased the number of radiation-induced S-phase specific chromosome aberrations and reduced the frequency of radiation-induced CtIP and Rad51 foci formation, suggesting that Torin2 works by blocking homologous recombination (HR)-mediated DNA repair resulting in an S-phase specific DNA repair defect. Accordingly, Torin2 reduced HR-mediated repair of I-Sce1-induced DNA damage and contributed to replication fork stalling. We conclude that radiosensitization of tumor cells by Torin2 is associated with disrupting ATR- and ATM-dependent DNA damage responses. Our findings support the concept of developing combination cancer therapies that incorporate ionizing radiation therapy and Torin2 or compounds with similar properties.

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The p53-binding protein 1 (53BP1) is a well-known DNA damage response (DDR) factor, which is recruited to nuclear structures at the site of DNA damage and forms readily visualized ionizing radiation (IR) induced foci. Depletion of 53BP1 results in cell cycle arrest in G2/M phase as well as genomic instability in human as well as mouse cells. Within the DNA damage response mechanism, 53BP1 is classified as an adaptor/mediator, required for processing of the DNA damage response signal and as a platform for recruitment of other repair factors. More recently, specific 53BP1 contributions to DSB repair pathway choice have been recognized and are being characterized. In this review, we have summarized recent advances in understanding the role of 53BP1 in regulating DNA DSBs repair pathway choice, variable diversity joining [V(D)J] recombination and class-switch recombination (CSR).

Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Chromosomes in PTEN deficient cells display both numerical as well as structural alterations including regional amplification. We found that PTEN deficient cells displayed a normal DNA damage response (DDR) as evidenced by the ionizing radiation (IR)-induced phosphorylation of Ataxia Telangiectasia Mutated (ATM) as well as its effectors. PTEN deficient cells also had no defect in Rad51 expression or DNA damage repair kinetics post irradiation. In contrast, caffeine treatment specifically increased IR-induced chromosome aberrations and mitotic index only in cells with PTEN, and not in cells deficient for PTEN, suggesting that their checkpoints were defective. Furthermore, PTEN-deficient cells were unable to maintain active spindle checkpoint after taxol treatment. Genomic instability in PTEN deficient cells could not be attributed to lack of PTEN at centromeres, since no interaction was detected between centromeric DNA and PTEN in wild type cells. These results indicate that PTEN deficiency alters multiple cell cycle checkpoints possibly leaving less time for DNA damage repair and/or chromosome segregation as evidenced by the increased structural as well as numerical alterations seen in PTEN deficient cells.

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.

The Mre11-Rad50-Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Organisms with sexual reproduction have two homologous copies of each chromosome. Meiosis is characterized by two successive cell divisions that result in four haploid sperms or eggs, each carrying a single copy of homologous chromosome. This process requires a coordinated reorganization of chromatin and a complex network of meiotic-specific signaling cascades. At the beginning of meiosis, each chromosome must recognize its homolog, then the two become intimately aligned along their entire lengths which allows the exchange of DNA strands between homologous sequences to generate genetic diversity. DNA double-strand breaks (DSBs) initiate meiotic recombination in a variety of organisms. Numerous studies have identified both the genomic loci of the initiating DSBs and the proteins involved in their formation. This review will summarize the activation and signaling networks required for the DSB response in meiosis.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

Heterochromatin protein 1β (HP1β), encoded by the Cbx1 gene, has been functionally linked to chromatin condensation, transcriptional regulation, and DNA damage repair. Here we report that testis-specific Cbx1 conditional knockout (Cbx1 cKO) impairs male germ cell development in mice. Depletion of HP1β negatively affected sperm maturation and increased seminiferous tubule degeneration in Cbx1 cKO mice. In addition, the spermatogonia have elevated γ-H2AX foci levels as do Cbx1 deficient mouse embryonic fibroblasts (MEFs) as compared to wild-type (WT) control MEFs. The increase in γ-H2AX foci in proliferating Cbx1 cKO cells indicates defective replication-dependent DNA damage repair. Depletion or loss of HP1β from human cells and MEFs increased DNA replication fork stalling and firing of new origins of replication, indicating defective DNA synthesis. Taken together, these results suggest that loss of HP1β in proliferating cells leads to DNA replication defects with associated DNA damage that impact spermatogenesis. [ABSTRACT FROM AUTHOR]

The DNA and protein complex known as chromatin is subject to posttranslational modifications (PTMs) that regulate cellular functions such that PTM dysregulation can lead to disease, including cancer. One critical PTM is acetylation/deacetylation, which is being investigated as a means to develop targeted cancer therapies. The histone acetyltransferase (HAT) family of proteins performs histone acetylation. In humans, MOF (hMOF), a member of the MYST family of HATs, acetylates histone H4 at lysine 16 (H4K16ac). MOF-mediated acetylation plays a critical role in the DNA damage response (DDR) and embryonic stem cell development. Functionally, MOF is found in two distinct complexes: NSL (nonspecific lethal) in humans and MSL (male-specific lethal) in flies. The NSL complex is also able to acetylate additional histone H4 sites. Dysregulation of MOF activity occurs in multiple cancers, including ovarian cancer, medulloblastoma, breast cancer, colorectal cancer, and lung cancer. Bioinformatics analysis of KAT8, the gene encoding hMOF, indicated that it is highly overexpressed in kidney tumors as part of a concerted gene coexpression program that can support high levels of chromosome segregation and cell proliferation. The linkage between MOF and tumor proliferation suggests that there are additional functions of MOF that remain to be discovered. [ABSTRACT FROM AUTHOR]

In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: 1) non-homologous end-joining (NHEJ), which re-ligates the broken ends of the DNA and 2) homologous recombination (HR), that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but posttranslational modification (PTMs) of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response (DDR). [ABSTRACT FROM AUTHOR]

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) de- ficiency. whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad5l foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad5l foci form equally well in the presence or absence of BRCA2, indicating that RadSZ can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells. [ABSTRACT FROM AUTHOR]

The molecular distinctions between mortality stages 1 (M1; senescence) and 2 (M2; crisis) of human replicative aging are ill defined. We demonstrate a qualitative difference between telomeric end associations at M1 and the end fusions that produce dicentric chromosomes and breakage-fusion cycles. Knockdown of ligase IV sufficient to completely inhibit radiation-induced dicentric chromosome formation had no effect on the frequency of telomere associations (TAs), establishing that TAs are not covalent conventional nonhomologous end-joining (NHEJ) products. TAs preceded and were more numerous than dicentric chromosomes. Cells initially tolerated dicentric chromosomes without dying, but eventually, a combination of too many TAs and dicentrics/complex chromosomal rearrangements resulted in apoptosis. We propose a working model in which end associations represent abortive DNA repair intermediates when the number of telomeric repeats is too small to completely inhibit DNA damage signaling but is sufficient to prevent the final covalent ligation step of NHEJ and induces the M1 checkpoint arrest in normal human cells. Rather than being all-or-none, telomere deprotection would thus proceed first through TAs before additional shortening leads to dicentric chromosomes. M2/crisis involves both qualitative changes (a shift from TAs to TAs plus dicentric chromosomes) and quantitative changes (an increase in the number of dysfunctional telomeres). [ABSTRACT FROM AUTHOR]

Packaging of the eukaryotic genome with histone and other proteins forms a chromatin structure that regulates the outcome of all DNA mediated processes. The cellular pathways that ensure genomic stability detect and repair DNA damage through mechanisms that are critically dependent upon chromatin structures established by histones and, particularly upon transient histone post-translational modifications. Though subjected to a range of modifications, histone methylation is especially crucial for DNA damage repair, as the methylated histones often form platforms for subsequent repair protein binding at damaged sites. In this review, we highlight and discuss how histone methylation impacts the maintenance of genome integrity through effects related to DNA repair and repair pathway choice. [ABSTRACT FROM AUTHOR]

Dot1/DOT1L (disruptor of telomeric silencing-1) is an evolutionarily conserved histone methyltransferase that methylates lysine 79 located within the globular domain of histone H3. Dot1 was initially identified by a genetic screen as a disruptor of telomeric silencing in Saccharomyces cerevisiae ; further, it is the only known non-SET domain containing histone methyltransferase. Methylation of H3K79 is involved in the regulation of telomeric silencing, cellular development, cell-cycle checkpoint, DNA repair, and regulation of transcription. hDot1L-mediated H3K79 methylation appears to have a crucial role in transformation as well as disease progression in leukemias involving several oncogenic fusion proteins. This review summarizes the multiple functions of Dot1/hDOT1L in a range of cellular processes. [ABSTRACT FROM AUTHOR]