Inactivation and activation of replication initiation factors

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A453-A454
• Main Author: Dutta, Anindya
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A453-d

The geminin‐Cdt1 balance is critical for preventing re‐replication in the same cell‐cycle. Besides neutralization by geminin, human Cdt1 is degraded at the onset of S phase and after DNA damage. We have shown that the there are two redundant pathways for degrading Cdt1: a Skp2 dependent pathway that requires phosphorylation of Cdt1 by cyclin dependent kinases and Cul4 dependent pathway that requires the interaction of Cdt1 with PCNA through a QXXVXXF motif. Both pathways are used for degrading Cdt1 in S phase. The Cul4, PCNA dependent pathway alone is used for degrading Cdt1 after DNA damage. Thus PCNA, known as a matchmaker for many proteins involved in DNA and chromatin metabolism, is also involved in the degradation of some of these proteins. Origins of replication are expected to recruit initiation proteins like ORC and Cdc6 in eukaryotes and provide a platform for unwinding DNA. We have tested whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated pre‐RC formation and replication initiation when fused to GAL4 DNA binding domain and recruited to plasmid DNA containing a tandem array of GAL4 binding sites. Replication occurred once per cell cycle and was inhibited by Geminin indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC‐Cdc6 complex and nascent strand abundance was highest near the GAL4 binding sites. Therefore the artificial origin recapitulates many of the regulatory features of physiological origins. Artificial recruitment of an eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene‐therapy.