K. and the cells subsequently arrest at a caffeine-sensitive G2/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated MK-8353 (SCH900353) histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability. The RecQ family of DNA helicases has been highly conserved MK-8353 (SCH900353) throughout the history of evolution from bacteria to humans (30, 32, 42). In bacteria and yeasts, there is a single family member in each case (RecQ in and mutants; in the latter case, this outcome is particularly evident in cells that have previously been exposed to hydroxyurea (HU), a ribonucleotide reductase inhibitor that arrests DNA replication (22, 57, 63). Although alterations in the rates at which genetic recombination events occur are characteristic of RecQ helicase mutants, it is widely thought that this abnormality is usually a downstream consequence of a primary defect in DNA replication (13, 46). Bacterial, yeast, and human RecQ helicase-deficient cells display abnormalities in DNA replication, which may manifest as an apparently unperturbed S phase or, more frequently, following UV irradiation-induced perturbation of replication, DNA replication inhibitors such as HU, or DNA-damaging drugs. Indeed, many RecQ helicase-deficient mutants are hypersensitive to one or more of these agents and display a defect in responding to replicational stress of this sort. For example, BS cell lines accumulate abnormally sized DNA replication intermediates and have a protracted S phase (39). mutants are hypersensitive to HU and show a defect specifically in S-phase checkpoint responses to both DNA damage and replication blockade (21). Similarly, mutants are HU sensitive and defective in recovery from an S-phase arrest: they are able to complete bulk DNA replication following release from the arrest, but they then undergo an aberrant mitosis (41, 57). The pattern of expression and subcellular localization of RecQ helicases are also suggestive of a role for these enzymes in DNA replication. Many MK-8353 (SCH900353) RecQ helicases, including BLM, MK-8353 (SCH900353) accumulate in cells only at or following progression through the G1/S transition in the cell division cycle (4, 34). Moreover, Sgs1p and BLM (as well as WRN) localize to sites of DNA replication either constitutively (particularly during late S phase) or more strikingly following perturbation of replication (21, 50, 55). These data suggest that there is a regulated translocation of RecQ helicase to sites of damaged or arrested replication forks in order to assist in restoration of DNA synthesis. Numerous models for the role(s) of RecQ helicases in replication fork repair have been proposed (30, 46, 60). These fall broadly into two categories that are not mutually exclusive: those that propose a role in the prevention of replication fork demise (for example, through the removal of aberrant DNA secondary structures from the template to easy passage of the replisome) and secondly, those that propose a role alongside the homologous recombination machinery in repairing collapsed or damaged forks. The conserved interactions between RecQ helicases and proteins required either for DNA replication, such as replication protein A (6, 7, 16), or for homologous recombination, such as RAD51 (64), are consistent with these proposed Rabbit Polyclonal to P2RY4 roles. One of the cellular responses to DNA damage and replication blockade is the activation of cell cycle checkpoints that serve to arrest cell cycle progression in order to allow time for the repair or bypass of.

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