Abstract
The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.
Highlights
flap endonuclease 1 (FEN1) Depletion and Transcription Inhibition Induce Replication Stress and a DNA Damage Response—Because telomeres are transcribed to produce telomeric repeat-containing RNA (TERRA) [31, 32], and because interference between replication and transcription is a known cause of genomic instability [5, 33, 34], we turned our attention to the impact that putative collisions between the replication and transcription machinery would have on telomere stability
Co-directional collisions are resolved by a mechanism that leaves behind an RNA:DNA hybrid/flap structure [20], which would need to be resolved in a eukaryotic cell to avoid a DNA damage response (DDR) and genomic instability [21, 22]
We have identified transcription as an important contributor to telomere fragility, and we have shown that FEN1 may resolve the RNA:DNA hybrid/flap structures resulting from collisions between the transcription and replication machinery
Summary
We show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data support a model in which co-directional replisome-RNAP collisions on the leading strand-replicated telomere produce RNA:DNA hybrid/flap structures that accumulate in the absence of FEN1. We propose that FEN1, a classical lagging strand replication protein, acts on the leading strand during telomere replication to resolve RNA:DNA hybrid/flap structures resembling Okazaki fragment substrates; in the absence of this activity, the subsequent replication stress and DNA damage manifest as telomere fragility. We believe this to be the first report placing an Okazaki fragment-processing protein explicitly on the leading strand during DNA replication
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