The role of Rad51 in an unperturbed cell cycle has been difficult to dissect from its DNA repair function. Here, using electron microscopy (EM) to visualize replication intermediates (RIs) assembled in Xenopus laevis egg extract we show that Rad51 is required to prevent the accumulation of ssDNA gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading and lagging strand DNA synthesis. Instead, ssDNA gaps behind forks, which are exacerbated on damaged templates, result from Mre11 dependent degradation of newly synthesized DNA strands as they can be suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct and unanticipated roles for Rad51 at replication forks demonstrating that Rad51 protects newly synthesised DNA from Mre11 dependent degradation and promotes continuous DNA synthesis.

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.

SUMMARY To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.

Changes in hippocampal function seem critical for cognitive impairment in Alzheimer's disease (AD). Although there is eventual loss of synapses in both AD and animal models of AD, deficits in spatial memory and inhibition of long-term potentiation (LTP) precede morphological alterations in the models, suggesting earlier biochemical changes in the disease. In the studies reported here we demonstrate that amyloid ␤-peptide (A␤) treatment of cultured hippocampal neurons leads to the inactivation of protein kinase A (PKA) and persistence of its regulatory subunit PKAII␣. Consistent with this, CREB phosphorylation in response to glutamate is decreased, and the decrease is reversed by rolipram, a phosphodiesterase inhibitor that raises cAMP and leads to the dissociation of the PKA catalytic and regulatory subunits. It is likely that a similar mechanism underlies 〈␤ inhibition of LTP, because rolipram and forskolin, agents that enhance the cAMP-signaling pathway, can reverse this inhibition. This reversal is blocked by H89, an inhibitor of PKA. These observations suggest that 〈␤ acts directly on the pathways involved in the formation of late LTP and agents that enhance the cAMP͞PKA͞CREB-signaling pathway have potential for the treatment of AD. A lzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by mild cognitive impairment at its onset and deficits in multiple cortical functions in later stages. To date, the vast majority of its symptoms have been attributed to the loss of synapses and the death of neurons that occur in the course of the disease. The overproduction and accumulation of the amyloid ␤-peptide (A␤) and particularly its 42-aa form (A␤ 1-42 ) have been shown to play a crucial role in both of these processes in animal models of AD (1, 2). Although these phenomena can account for the late debilitating stages of the disease, the mechanisms by which A␤ causes early cognitive and behavioral changes remain a matter of conjecture. Recent studies on animal models of AD have highlighted the discrepancy between behavioral deficits and neuropathological findings. Electrophysiological studies on mice that overexpress A␤ show impairment of long-term potentiation (LTP) that does not correlate with the extent of synaptic loss, amyloid deposition, or cell death (3-5). In addition, animals without detectable accumulation of A␤ have been reported to have behavioral deficits (6, 7). While examining gene expression in nerve growth factorprimed PC12 cells that had been exposed to A␤ 1-42 for 3 h, we observed that a group of genes including CREB2 (ATF4) and ubiquitin C-terminal hydrolase, which have been implicated in the switch from early to late LTP, were regulated in a manner consistent with an 〈␤-mediated inhibition of the cAMPmediated signaling pathway for the consolidation of LTP. ʈ The details of the biochemical pathway mediating the switch from early to late LTP have been worked out in aplysia and mice (8) and depend on the activation of the transcription factor CREB by phosphorylat...