Program > Browse abstracts by author > Schofield Phil

Long interspersed element 1 (LINE-1, L1) is the only active retrotransposon in modern humans and its aberrant overexpression is found in nearly half of human cancers. L1 generates de novo insertions of itself via a ‘copy-and-paste' mechanism that is dependent on two protein-coding open reading frames (ORFs): ORF1p, an RNA-binding protein and ORF2p, an endonuclease (EN) and reverse transcriptase (RT). Accumulating evidence implies that the insertion of L1 into the genome occurs during S phase and that DNA replication and replication-coupled repair factors limit retrotransposition. Additionally, L1 expression generates molecular vulnerabilities to the loss of DNA replication and repair factors. These synthetic lethal interactions suggest a model wherein L1 retrotransposition intermediates hinder DNA replication progression, a condition referred to as DNA replication stress (RS), that can lead to fork collapse, DNA damage, and consequent genomic instability. To explore this model and impact of L1 expression on replication fork dynamics, we performed DNA fiber assays and found L1-expressing cells display a marked reduction in replication fork velocity accompanied with an asymmetric pattern of sister replication tracts, an indication of direct fork stalling. Intriguingly, we observed higher frequency of newly fired DNA replication origins in L1-expressing cells. We found that inhibition of new origin firing using CDC7 inhibitors completely rescued L1-induced RS, suggesting its dependence on aberrant origin. Mitotic entry with persistent replication intermediates through S/G2 checkpoint triggers a mitotic DNA synthesis (MiDAS) and compromises chromosome segregation, causing chromosomal breakage and genome instability. Indeed, we found higher frequencies of MiDAS and cytological markers of chromosome missegregation upon L1 expression. We are currently testing the hypothesis that aberrant origin firing underlies L1-induced replication stress and chromosome instability. Our findings are expected to reveal the molecular mechanisms underlying L1-associated DNA damage and genome instability, a hallmark of cancers.