Continued primer synthesis at stalled replication forks contributes to checkpoint activation

• Published in: The Journal of cell biology 2010-04, Vol.189 (2), p.233-246
• Main Authors: Van, Christopher, Yan, Shan, Michael, W. Matthew, Waga, Shou, Cimprich, Karlene A.
• Format: Article
• Language: English
• Subjects: Animals; Antigens; Aphidicolin - metabolism; Ataxia Telangiectasia Mutated Proteins; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cells; Cellular biology; Checkpoint Kinase 1; Chromatin; Chromatin - metabolism; Deoxyribonucleic acid; DNA; DNA damage; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; DNA Primers - genetics; DNA Primers - metabolism; DNA Replication; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA-Binding Proteins; Eggs; Enzyme Inhibitors - metabolism; Eukaryotes; Female; Frogs; Gels; Male; Phosphorylation; Plasmids; Protein Kinases - genetics; Protein Kinases - metabolism; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Protein synthesis; Spermatozoa; Xenopus laevis - genetics; Xenopus laevis - metabolism; Xenopus Proteins - genetics; Xenopus Proteins - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200909105

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-δ, and Pol-ε, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer—template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.