PARP2 controls double-strand break repair pathway choice by limiting 53BP1 accumulation at DNA damage sites and promoting end-resection.

Double strand breaks (DSBs) are one of the most toxic lesions to cells. DSB repair by the canonical non-homologous end-joining (C-EJ) pathway involves minor, if any, processing of the broken DNA-ends, whereas the initiation of DNA resection channels the broken-ends toward DNA repair pathways using various lengths of homology. Mechanisms that control the resection initiation are thus central to the regulation to the choice of DSB repair pathway. Therefore, understanding the mechanisms which regulate the initiation of DNA end-resection is of prime importance. Our findings reveal that poly(ADP-ribose) polymerase 2 (PARP2) is involved in DSBR pathway choice independently of its PAR synthesis activity. We show that PARP2 favors repair by homologous recombination (HR), single strand annealing (SSA) and alternative-end joining (A-EJ) rather than the C-EJ pathway and increases the deletion sizes at A-EJ junctions. We demonstrate that PARP2 specifically limits the accumulation of the resection barrier factor 53BP1 at DNA damage sites, allowing efficient CtIP-dependent DNA end-resection. Collectively, we have identified a new PARP2 function, independent of its PAR synthesis activity, which directs DSBs toward resection-dependent repair pathways.

PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels.

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells' poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

Variations in the mRNA expression of poly(ADP-ribose) polymerases, poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome.

In order to assess the variation in expression of poly(ADP-ribose) polymerase (PARP) family members and the hydrolases that degrade the poly(ADP-ribose) polymers they generate and possible associations with classical pathological parameters, including long-term outcome, the mRNA levels of PARP1, PARP2, PARP3, poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3) were examined using quantitative reverse transcription polymerase chain reaction in 443 unilateral invasive breast cancers and linked to hormonal status, tumor proliferation and clinical outcome. PARP1 mRNA levels were the highest among these five genes in both normal and tumor tissues, with a 2.45-fold higher median level in tumors compared to normal tissues. Tumors (34.1%) showed PARP1 overexpression (>3 fold relative to normal breast tissues) compared to underexpression (<0.33 fold) in only 0.5%. This overexpression was seen in all breast tumor subgroups, with the highest fraction (51%) seen in the HR-positive/ERBB2-positive subgroup and was not highly associated with any other classical predictive factors. No correlation was seen between PARP1 mRNA and PARP-1 protein levels in a subset of 31 tumors. PARP3 was underexpressed in 10.4% of tumors, more frequently in the HR-negative tumors (25.4%) than the HR-positive tumors (5.9%). This PARP3 underexpression was mutually exclusive with a PARP1 overexpression. PARP2 levels were unchanged between normal and tumor tissues and few tumors showed overexpression of PARG (3.8%) or ARH3 (3.4%). Within the subgroup of triple negative tumors, PARG mRNA levels below the median were associated with a higher risk of developing metastases (p = 0.039) raising the possibility this might be marker of clinical outcome.

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells.

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation.

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.

Recombination and the Tel1 and Mec1 checkpoints differentially effect genome rearrangements driven by telomere dysfunction in yeast.

In telomerase-deficient Saccharomyces cerevisiae, telomeres are maintained by recombination. Here we used a S. cerevisiae assay for characterizing gross chromosomal rearrangements (GCRs) to analyze genome instability in post-senescent telomerase-deficient cells. Telomerase-deficient tlc1 and est2 mutants did not have increased GCR rates, but their telomeres could be joined to other DNAs resulting in chromosome fusions. Inactivation of Tel1 or either the Rad51 or Rad59 recombination pathways in telomerase-deficient cells increased the GCR rate, even though telomeres were maintained. The GCRs were translocations and chromosome fusions formed by nonhomologous end joining. We observed chromosome fusions only in mutant strains expressing Rad51 and Rad55 or when Tel1 was inactivated. In contrast, inactivation of Mec1 resulted in more inversion translocations such as the isochromosomes seen in human tumors. These inversion translocations seemed to be formed by recombination after replication of broken chromosomes.

Protocol for automated multivariate quantitative-image-based cytometry analysis by fluorescence microscopy of asynchronous adherent cells.

Here, we present a protocol for multivariate quantitative-image-based cytometry (QIBC) analysis by fluorescence microscopy of asynchronous adherent cells. We describe steps for the preparation, treatment, and fixation of cells, sample staining, and imaging for QIBC. We then detail image analysis with our open source Fiji script developed for QIBC and present multiparametric data visualization. Our QIBC Fiji script integrates modern artificial-intelligence-based tools, applying deep learning, for robust automated nuclei segmentation with minimal user adjustments, a major asset for efficient QIBC analysis. For complete details on the use and execution of this protocol, please refer to Besse et al. (2023).1.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype.

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.

Analysis of gross-chromosomal rearrangements in Saccharomyces cerevisiae.

Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA.

Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.

Stabilization of dicentric translocations through secondary rearrangements mediated by multiple mechanisms in S. cerevisiae.

BACKGROUND: The gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instability METHODOLOGY/PRINCIPAL FINDINGS: The structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR. CONCLUSIONS/SIGNIFICANCE: HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.

Chromosome healing by de novo telomere addition in Saccharomyces cerevisiae.

The repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerase-dependent addition of telomeres at non-telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA-template (guide RNA) at short regions of homology is followed by extension of the nascent 3'-end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single-stranded DNA-binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing-extension-dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae.

Chromosome healing through terminal deletions generated by de novo telomere additions in Saccharomyces cerevisiae.

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3' end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a low-complexity guide RNA sequence.

Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression.

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1.

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability.

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.