Short CCG repeat in huntingtin gene is an obstacle for replicative DNA polymerases, potentially hampering progression of replication fork.
Trinucleotide repeats (TNRs) are highly unstable in genomes, and their expansions are linked to human disorders. DNA replication is reported to be involved in TNR instability, but the current models are insufficient in explaining TNR expansion is induced during replication. Here, we investigated replication fork progression across huntingtin (HTT)-gene-derived fragments using an Escherichia coli oriC plasmid DNA replication system. We found most of the forks to travel smoothly across the HTT fragments even when the fragments had a pathological length of CAG/CTG repeats (approximately 120 repeats). A little fork stalling in the fragments was observed, but it occurred within a short 3'-flanking region downstream of the repeats. This region contains another short TNR, (CCG/CGG)7 , and the sense strand containing CCG repeats appeared to impede the replicative DNA polymerase Pol III. Examining the behavior of the human leading and lagging replicative polymerases Pol epsilon (hPolε) and Pol delta (hPolδ) on this sequence, we found hPolδ replicating DNA across the CCG repeats but hPolε stalling at the CCG repeats even if the secondary structure is eliminated by a single-stranded binding protein. These findings offer insights into the distinct behavior of leading and lagging polymerases at CCG/CGG repeats, which may be important for understanding the process of replication arrest and genome instability at the HTT gene.
Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Nara, Japan.
Hang Phuong
HP
Le
Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.|Department of Toxicogenomics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Yuji
Y
Masuda
Department of Biology, School of Sciences, Kyushu University, Fukuoka, 812-8581, Japan.
Toshiki
T
Tsurimoto
Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Nara, Japan.
Satoko
S
Maki
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
Tsutomu
T
Katayama
Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Nara, Japan.
Asako
A
Furukohri
Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Nara, Japan.
Hisaji
H
Maki
D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071058:Huntingtin Protein; D009097:Multienzyme Complexes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D018911:Trinucleotide Repeats; D000099272:Replisomes
D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D000071058:Huntingtin Protein; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D000099272:Replisomes; C086055:HTT protein, human