Interactions Among DNA Ligase I, the Flap Endonuclease and Proliferating Cell Nuclear Antigen in the Expansion and Contraction of CAG Repeat Tracts in Yeast

Abstract

My aim is to agitate and disturb people. I'm not selling bread, I'm selling yeast.Miguel de Unamuno ABSTRACT Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele pol30-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.

CTG/CAG Repeat Instability Is Modulated by the Levels of Human DNA Ligase I and Its Interaction with Proliferating Cell Nuclear Antigen

“…On several points our results agree with previous yeast studies (30,31) and extend them by revealing specific contributions of replication-or post-replication repair to repeat instability; (i) mutant low activity ligase did not affect instability, (ii) overexpression of wt-LigI increased instability, and (iii) a dependence upon PCNA interaction was observed in both studies. Although the yeast study observed a replication direction dependence on these effects, we observed effects for both directions.…”

“…(i) In yeast, abolishing the interaction between LigI and PCNA increased TNR instability levels (30). Likewise, overexpression of yeast LigI also increased repeat instability.…”

C-Terminal Flap Endonuclease (rad27) Mutations: Lethal Interactions With a DNA Ligase I Mutation (cdc9-p) and Suppression by Proliferating Cell Nuclear Antigen (POL30) in Saccharomyces cerevisiae

“…The PIP box fits into the interdomain connector loop (IDCL) of PCNA to provide a protein-protein interaction surface (Gomes and Burgers 2000;Chapados et al 2004;Sakurai et al 2005;Pascal et al 2006). Mutations in the PIP box or the IDCL that impair the interaction of DNA ligase and the flap endonuclease to PCNA lead to genomic instability (Amin and Holm 1996;Eissenberg et al 1997;Gary et al 1999;Refsland and Livingston 2005;Subramanian et al 2005). We have reported that the double mutants made by combinations of cdc9-p, rad27-p, and pol30-90-mutations with alterations of the PIP box or the IDCL in the respective proteins-have synergistic phenotypes with respect to MMS sensitivity and to trinucleotide repeat instability (Refsland and Livingston 2005).…”

“…Mutations in the PIP box or the IDCL that impair the interaction of DNA ligase and the flap endonuclease to PCNA lead to genomic instability (Amin and Holm 1996;Eissenberg et al 1997;Gary et al 1999;Refsland and Livingston 2005;Subramanian et al 2005). We have reported that the double mutants made by combinations of cdc9-p, rad27-p, and pol30-90-mutations with alterations of the PIP box or the IDCL in the respective proteins-have synergistic phenotypes with respect to MMS sensitivity and to trinucleotide repeat instability (Refsland and Livingston 2005). These results suggest that the two enzymes function in a concerted manner that is facilitated by PCNA.…”

“…To further elucidate how the interaction of DNA ligase with PCNA is ordered, we performed a genetic screen to identify mutations that are synthetically lethal with cdc9-p (F44A/F35A), an allele of DNA ligase that has impaired binding to PCNA (Refsland and Livingston 2005;Subramanian et al 2005). We postulated that genes recovered from this screen would function in DNA repair, replication, and recombination or would be involved in ordering the DNA ligase-PCNA interaction.…”