Poly(ADP-ribosyl)gycohydrolase (PARG) prevents the accumulation of unusual replication structures during unperturbed S phase

Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribose) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibition per se slows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and ssDNA gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG defective cells displayed both ATM and ATR activation, as well as chromatin recruitment of standard double strand break repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restart are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy

Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates

Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a crucial anticancer barrier. DDR inactivation in these conditions promotes genome instability and tumor progression, but the underlying molecular mechanisms are elusive. We found that overexpression of both Cyclin E and Cdc25A rapidly slowed down replication forks and induced fork reversal, suggestive of increased topological stress. Surprisingly, these phenotypes, per se, are neither associated with chromosomal breakage nor with significant DDR activation. Oncogene-induced DNA breakage and DDR activation instead occurred upon persistent G2/M arrest or, in a checkpoint-defective context, upon premature CDK1 activation. Depletion of MUS81, a cell cycle-regulated nuclease, markedly limited chromosomal breakage and led to further accumulation of reversed forks. We propose that nucleolytic processing of unusual replication intermediates mediates oncogene-induced genotoxicity and that limiting such processing to mitosis is a central anti-tumorigenic function of the DNA damage checkpoints

Revista escolar La Pizarra

El Centro edita una revista llamada 'La Pizarra', en la que se recogen los trabajos, actividades e inquietudes de todos los miembros de la comunidad escolar: padres, maestros y niños. Con la edición de esta revista se pretende alcanzar los siguientes objetivos: Que los niños adquieran ciertos conocimientos relacionados con la prensa (maquetación, ilustraciones etc.); Promover en los alumnos conductas de interés hacia la lectura; Fomentar el intercambio de noticias entre distintos ciclos; Aprender a coordinar las diferentes noticias que llegan al equipo de redacción; Comprender como surgen las noticias y como se elaboran; Entender que la revista debe de tener distintas secciones para que se cubran diferentes campos de interés y atraiga a diversos tipos de lectores; Darse cuenta de que cada sección tiene sus características diferentes a la hora de confeccionarla y tratarla; Ser conscientes de que la edición de una revista es el resultado del trabajo coordinado de un equipo; Comparar diferentes revistas de tirada nacional y ver a quién van dirigidas; Crear por medio de una revista un Centro motivador y de unión entre las distintas clases del Centro; Conseguir que la revista sea un escaparate hacia el exterior del Centro; Fomentar la participación de los distintos sectores de la comunidad; Hacer de la escritura propia una fuente de intercambio de ideas..Gobierno de Cantabria. Consejeria de Educación y JuventudCantabriaES

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks

A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy