title: DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene.
abstract: DNA has the ability to form a variety of secondary structures in addition to the normal B-form DNA, including hairpins and quadruplexes. These structures are implicated in a number of neurological diseases and cancer. Expansion of a GGGGCC repeat located at C9orf72 is associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. This repeat expands from two to 24 copies in normal individuals to several hundreds or thousands of repeats in individuals with the disease. Biochemical studies have demonstrated that as little as four repeats have the ability to form a stable DNA secondary structure known as a G-quadruplex. Quadruplex structures have the ability to disrupt normal DNA processes such as DNA replication and transcription. Here we examine the role of GGGGCC repeat length and orientation on DNA replication using an SV40 replication system in human cells. Replication through GGGGCC repeats leads to a decrease in overall replication efficiency and an increase in instability in a length-dependent manner. Both repeat expansions and contractions are observed, and replication orientation is found to influence the propensity for expansions or contractions. The presence of replication stress, such as low-dose aphidicolin, diminishes replication efficiency but has no effect on instability. Two-dimensional gel electrophoresis analysis demonstrates a replication stall with as few as 20 GGGGCC repeats. These results suggest that replication of the GGGGCC repeat at C9orf72 is perturbed by the presence of expanded repeats, which has the potential to result in further expansion, leading to disease.
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Ryan Griffin
authors.initials: RG
authors.lastname: Thys
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908 yw4b@virginia.edu.
authors.forename: Yuh-Hwa
authors.initials: YH
authors.lastname: Wang
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000073885:C9orf72 Protein; D004261:DNA Replication; D054856:G-Quadruplexes; D057809:HEK293 Cells; D006801:Humans; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000502:physiology; D014779:Virus Replication / Q000502:physiology
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D011506:Proteins
keywords: DNA; DNA replication; DNA structure; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); genomic instability