CTG/CAG repeat instability is modulated by the levels of human DNA ligase I and its interaction with proliferating cell nuclear antigen: a distinction between replication and slipped-DNA repair.
Mechanisms contributing to disease-associated trinucleotide repeat instability are poorly understood. DNA ligation is an essential step common to replication and repair, both potential sources of repeat instability. Using derivatives of DNA ligase I (hLigI)-deficient human cells (46BR.1G1), we assessed the effect of hLigI activity, overexpression, and its interaction with proliferating cell nuclear antigen (PCNA) upon the ability to replicate and repair trinucleotide repeats. Compared with LigI(+/+), replication progression through repeats was poor, and repair tracts were broadened beyond the slipped-repeat for all mutant extracts. Increased repeat instability was linked only to hLigI overexpression and expression of a mutant hLigI incapable of interacting with PCNA. The endogenous mutant version of hLigI with reduced ligation activity did not alter instability. We distinguished the DNA processes through which hLigI contributes to trinucleotide instability. The highest levels of repeat instability were observed under the hLigI overexpression and were linked to reduced slipped-DNAs repair efficiencies. Therefore, the replication-mediated instability can partly be attributed to errors during replication but also to the poor repair of slipped-DNAs formed during this process. However, repair efficiencies were unaffected by expression of a PCNA interaction mutant of hLigI, limiting this instability to the replication process. The addition of purified proteins suggests that disruption of LigI and PCNA interactions influences trinucleotide repeat instability. The variable levels of age- and tissue-specific trinucleotide repeat instability observed in myotonic dystrophy patients and transgenic mice may be influenced by varying steady state levels of DNA ligase I in these tissues and during different developmental windows.
López Castel
Arturo
A
Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.
Tomkinson
Alan E
AE
Pearson
Christopher E
CE
D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014162:Transfection; D018911:Trinucleotide Repeats / Q000235:genetics*
C000607892:LIG1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP