Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability.

R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

The accuracy of pedicle screw placement using intraoperative image guidance systems.

OBJECT: Several retrospective studies have demonstrated higher accuracy rates and increased safety for navigated pedicle screw placement than for free-hand techniques; however, the accuracy differences between navigation systems has not been extensively studied. In some instances, 3D fluoroscopic navigation methods have been reported to not be more accurate than 2D navigation methods for pedicle screw placement. The authors of this study endeavored to identify if 3D fluoroscopic navigation methods resulted in a higher placement accuracy of pedicle screws. METHODS: A systematic analysis was conducted to examine pedicle screw insertion accuracy based on the use of 2D, 3D, and conventional fluoroscopic image guidance systems. A PubMed and MEDLINE database search was conducted to review the published literature that focused on the accuracy of pedicle screw placement using intraoperative, real-time fluoroscopic image guidance in spine fusion surgeries. The pedicle screw accuracy rates were segregated according to spinal level because each spinal region has individual anatomical and morphological variations. Descriptive statistics were used to compare the pedicle screw insertion accuracy rate differences among the navigation methods. RESULTS: A total of 30 studies were included in the analysis. The data were abstracted and analyzed for the following groups: 12 data sets that used conventional fluoroscopy, 8 data sets that used 2D fluoroscopic navigation, and 20 data sets that used 3D fluoroscopic navigation. These studies included 1973 patients in whom 9310 pedicle screws were inserted. With conventional fluoroscopy, 2532 of 3719 screws were inserted accurately (68.1% accuracy); with 2D fluoroscopic navigation, 1031 of 1223 screws were inserted accurately (84.3% accuracy); and with 3D fluoroscopic navigation, 4170 of 4368 screws were inserted accurately (95.5% accuracy). The accuracy rates when 3D was compared with 2D fluoroscopic navigation were also consistently higher throughout all individual spinal levels. CONCLUSIONS: Three-dimensional fluoroscopic image guidance systems demonstrated a significantly higher pedicle screw placement accuracy than conventional fluoroscopy or 2D fluoroscopic image guidance methods.

NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies.

Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

The ATR pathway: fine-tuning the fork.

The proper detection and repair of DNA damage is essential to the maintenance of genomic stability. The genome is particularly vulnerable during DNA replication, when endogenous and exogenous events can hinder replication fork progression. Stalled replication forks can fold into deleterious conformations and are also unstable structures that are prone to collapse or break. These events can lead to inappropriate processing of the DNA, ultimately resulting in genomic instability, chromosomal alterations and cancer. To cope with stalled replication forks, the cell relies on the replication checkpoint to block cell cycle progression, downregulate origin firing, stabilize the fork itself, and restart replication. The ATR (ATM and Rad3-related) kinase and its downstream effector kinase, Chk1, are central regulators of the replication checkpoint. Loss of these checkpoint proteins causes replication fork collapse and chromosomal rearrangements which may ultimately predispose affected individuals to cancer. This review summarizes our current understanding of how the ATR pathway recognizes and stabilizes stalled replication forks.