Cell unable to complete the recombinational repair process show abnormalities at cell division.

Stephen West : Senataxin and the maintenance of genome stability in neuronal cells

Introduction

Neuronal tissues are exposed to high levels of oxidative stress and have low levels of antioxidant enzymes, making them particularly sensitive to defects in DNA repair.

Defective cells by M-fish showing several genetic abnormalities.

Figure 1: Comparison of normal (left) and AOA-2 defective cells by M-fish showing several genetic abnormalities (chromosome duplication/loss and breakage).

Due to their limited capacity for self-renewal, unrepaired lesions can accumulate over a period of years, potentially blocking transcription by RNA polymerase II (RNAPII), and trigger cell death either by progressively depriving the cell of vital transcripts or through apoptosis.

Neuronal tissues are exposed to high levels of oxidative stress and have low levels of antioxidant enzymes, making them particularly sensitive to defects in DNA repair. Due to their limited capacity for self-renewal, unrepaired lesions can accumulate over a period of years, potentially blocking transcription by RNA polymerase II (RNAPII), and trigger cell death either by progressively depriving the cell of vital transcripts or through apoptosis. As a consequence, genome instability in the adult brain leads to impaired neural development and neurodegeneration.

We are interested in understanding the molecular defects that lead to the neurodegenerative syndromes collectively known as Ataxia with Oculomotor Apraxia (AOA). These syndromes are subdivided into a number of sub-classes: we are particularly interested in AOA1, caused by mutations in APTX, and AOA2 caused by mutations in SETX. Other neurological diseases, including Amyotrophic Lateral Sclerosis 4 (ALS4), Tremor-Ataxia Syndrome (TAS), Autosomal Dominant proximal Spinal Muscular Atrophy (ADSMA) and Charcot-Marie-Tooth (CMT) disease have also been linked with mutations in the SETX gene.

Previously, we discovered that the product of the APTX gene, Aprataxin, is a novel DNA-repair protein that resolves abortive DNA ligation intermediates by catalysing the removal of 5'-DNA adenylates that form when DNA ligases attempt to rejoin 'dirty' breaks caused by oxidative DNA damage. The protein also acts upon non-ligatable RNA-DNA junctions.

We are now determining the cellular role of Senataxin, the product of SETX, and why mutations in this gene cause AOA2. Senataxin, is a 2,677 amino acid protein that contains an RNA/DNA helicase superfamily I domain. Although the molecular functions of Senataxin, and how mutations therein lead to neuropathy remain unknown, this putative RNA/DNA helicase is considered to be an important player in the resolution of RNA/DNA hybrids (R-loops) formed during transcription termination or the RNA-DNA damage response (RDDR).

Previous studies have shown that SETX localizes to sites of collision between components of the replisome and the transcription apparatus and that it is targeted to R-loops, where it plays an important role at the interface of transcription and RDDR. Accumulating evidence indicates that R-loops may be an important source of replication stress-induced tumourigenesis. We find that loss of SETX in both human and mouse cells causes hypersensitivity to treatment with agents that cause either replication stress or induce the formation of R-loops. Furthermore, SETX deficiency promotes the formation of replication stress-induced genomic instability and chromosomal rearrangements.

We are now using genomic approaches to determine whether loss of SETX results in altered gene expression, differential methylation patterns or copy number alterations at replication stress hotspots.