Anil Ganesh

Repair and misrepair of site-specific DNA double-strand breaks by human cell extracts.

The rejoining by human cell extracts of a double-strand break induced by endonuclease treatment at one of several sites within a small DNA molecule was studied. Rejoining was found at each of 8 sites tested, but the rejoin efficiency varied with the nature of the break (e.g., breaks with cohesive ends were rejoined more efficiently than blunt-ended breaks). Extracts from primary and immortalized cell lines, as well as those from individuals with ataxia telangiectasia (A-T), showed the same pattern of relative rejoin efficiencies. However, mis-rejoining varied with the cell extract used, and was particularly elevated with two immortalized A-T cell lines. Mixing experiments showed that the mis-rejoining property of extracts could act in a semi-dominant fashion, depending on the individual efficiencies of the component extracts. The mis-rejoin mechanism involved deletion at sites of short direct repeats at various distances from the initial break site. A model of deletion formation (the strand-exposure and repair model) is restated to explain the sequence repeat dependence found, and is compared to models of homologous DNA recombination.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.

Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Defining the processes that sustain telomere maintenance is critical to our understanding of cancer and longevity. PIF1 is a nonprocessive 5->3 human DNA helicase that exhibits broad substrate specificity. In vitro studies have implicated PIF1 in maintaining telomeres and processing stalled DNA replication forks, but disruption of the murine Pif1 gene did not yield any apparent phenotype. In this study, we evaluated the function of the PIF1 gene in human cells by using siRNA knockdown strategies to gauge its role in the response to DNA replication stress. We found that PIF1 depletion reduced the survival of both p53-deficient and p53-proficient human tumor cells by triggering apoptosis. In contrast, nonmalignant cells were unaffected by PIF1 depletion. Apoptosis induction in tumor cells was augmented by cotreatment with replication inhibitors (thymidine, hydroxyurea, or gemcitabine). When sensitive PIF1-depleted cells were released from a thymidine-induced S-phase arrest, there remained a subpopulation of cells that failed to enter S-phase. This cell subpopulation displayed an increase in levels of cyclin E and p21, as well as a deficiency in S-phase checkpoint markers that were induced with thymidine in PIF1 expressing cells. Specifically, CHK1 activation was suppressed and we detected no consistent changes in ATM S1981 autophosphorylation, γH2AX induction, or RPA hyperphosphorylation. Death in PIF1-depleted cells was detected in late G(1)/early S-phase and was dependent on caspase-3 activity. Taken together, our findings suggest roles for PIF1 in S-phase entry and progression that are essential to protect human tumor cells from apoptosis.

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.

MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response

Summary Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP’s role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.

Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells

The replication protein A (RPA)–ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.

Mechanisms of Resistance to Ionising Radiations: Genetic and Molecular Studies on Ataxia-Telangiectasia and Related Radiation-Sensitive Mutants

In mammalian cells the mechanisms of resistance to damage caused by ionising radiations (IR) are largely unknown. Knowledge of the types of damage induced and of the enzymes, identified mostly in lower organisms, which act upon that damage may suggest that certain mechanisms are likely to prevail. However, in this respect, the diversity of damage caused by IR in the genetic material (and presumably in some other cellular molecules) leads to some uncertainty; a large number of different types of altered chemical products have been identified in irradiated DNA (Hutchinson, 1985; Teoule, 1987). This suggests that a large number of enzymes may be involved in recovery from IR damage, although some will be more important than others when considering a given cellular response. IR-induced cell killing, for example, has been linked to the production of DNA strand breakage, especially double-strand breaks (dsb). In E. coli there are a number of genes affecting in the repair of dsb; mutations in these genes lead to a reduction in the efficiency of dsb rejoining to varying extents and to enhanced sensitivity to IR (e.g., Sargentini and Smith 1986). Probably the best example to illustrate this link is the use of an X-ray-sensitive yeast mutant rad54-3 which is also temperature-sensitive: it can repair dsb at 23¼ but not at 36°C and shows a quantitative recovery of radiation resistance as the temperature is shifted to allow dsb repair (Frankenberg-Schwager, et al., 1988). Additionally, in yeast it has been found that the induction of approximately one dsb by IR is equivalent to one lethal event in the absence of repair (Ho, 1975; Resnick and Martin, 1976; Frankenberg, et al., 1981). These analyses show that mutants sensitive to IR are potentially useful in defining mechanisms, although such mutants have to be carefully characterized before they can be of value.

The relationship of CDK18 expression in breast cancer to clinicopathological parameters and therapeutic response

Background Cyclin-Dependent Kinases (CDKs) are established anti-cancer drug targets and a new generation of CDK inhibitors are providing clinical benefits to a sub-set of breast cancer patients. We have recently shown that human CDK18 promotes efficient cellular responses to replication stress. In the current study, we have investigated the clinicopathological and functional significance of CDK18 expression levels in breast cancers. Results High CDK18 protein expression was associated with a triple negative and basal-like phenotype (p = 0.021 and 0.027 respectively) as well as improved patient survival, which was particularly significant in ER negative breast cancers (n = 594, Log Rank 6.724, p = 0.01) and those treated with chemotherapy (n = 270, Log Rank 4.575, p = 0.03). In agreement with these clinical findings, breast cancer cells genetically manipulated using a dCRISPR approach to express high levels of endogenous CDK18 exhibited an increased sensitivity to replication stress-inducing chemotherapeutic agents, as a consequence to defective replication stress signalling at the molecular level. Conclusions These data reveal that CDK18 protein levels may predict breast cancer disease progression and response to chemotherapy, and provide further rationale for potential targeting of CDK18 as part of novel anti-cancer strategies for human cancers. Materials and Methods CDK18 protein expression was evaluated in 1650 breast cancers and correlated to clinicopathological parameters and survival outcomes. Similar analyses were carried out for genetic and transcriptomic changes in CDK18 within several publically available breast cancer cohorts. Additionally, we used a deactivated CRISPR/Cas9 approach (dCRISPR) to elucidate the molecular consequences of heightened endogenous CDK18 expression within breast cancer cells.

Abstract B56: Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency.

Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly replication and genome stability in lower and higher eukaryotes. While mouse pif1-/- knockouts show no phenotype and PIF1 is dispensable in non-cancerous human cells, Pif1 knockdowns sensitize many tumor cell lines to replication inhibitors, slow S-phase progression, and trigger apoptosis. Here we investigated the effects of PIF1 depletion on a non-tumor cell line, MRC5-SV2, and HRAS-transformed derivatives. We show that PIF1 depletion reduces the growth and viability of RAS-transformed MRC5-SV2 cells relative to parental and increases sensitivity to the therapeutic DNA replication inhibitor gemcitabine. Since previous work suggested that PIF1 depletion affected S-phase entry and transition in cultured tumor cells, we used single fiber analysis of pulse-labeled DNA to investigate the potential role of PIF1 in these events. Here we show that PIF1 depletion triggers replication stress characterized by slower fork rates and increased fork arrest during normal cycling conditions in non-tumor MRC5-SV2 cells. These effects were enhanced in the RAS-transformed derivative. Impaired fork movement upon PIF1-depletion did not augment DNA double-stranded break formation or trigger DNA damage responses. However PIF1 depletion affected resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and S-phase entry. Taken together, our data establish a novel functional role for human PIF1 in DNA replication that becomes critical for cell growth during oncogenic stress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B56. Citation Format: Mary E. Gagou, Anil Ganesh, Mark Meuth. Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B56.