Roles of SLX complexes in DNA replication and repair
File(s)
Author(s)
Sebald, Marie
Type
Thesis
Abstract
Structure-selective endonucleases (SSEs) are essential for genome stability and often harbour tumour suppressor functions. SLX4 provides a scaffold for the assembly of three structure selective nucleases, SLX1, XPF-ERCC1 and MUS81-EME1, which cleave a variety of DNA secondary structures arising during recombination, replication, and repair. Apart from SSEs, SLX4 provides a binding platform for plethora of DNA damage response factors including the less well characterised SLX4 interacting protein (SLX4IP). Although SLX4IP has been shown to be a regulator of the DNA endonuclease complex SLX1-SLX4-XPF-ERCC1-MUS81-EME1 and helicases (BLM) at recombining telomeres, little is known about its importance in DNA repair beyond telomere maintenance.
In this work, using a genome wide CRISPR-Cas9 screen, SLX4IP loss was identified as synthetic lethal with loss of the Okazaki fragment maturation protein flap endonuclease 1 (FEN1). Synthetic lethality was due to defects in DNA replication, defined by an increase in single-stranded DNA (ssDNA) gap accumulation behind replication forks and elevated S-phase specific ADP-ribosylation, a hallmark for defects in Okazaki fragment maturation. SLX4IP is shown to target the SLX1-SLX4-XPF-ERCC1 nuclease complex to active replication forks upon loss of FEN1 to process lagging strand intermediates. SLX1 and XPF were further identified as the mediators of the lethality in FEN1 KO cells. Processing of lagging strand intermediates via SLX4IP in combination with SLX1-SLX4-XPF-ERCC1 provides an alternative mechanism to preserve lagging strand integrity. Together, these findings deliver novel insights into the contribution of structure-selective endonucleases and associated proteins to genomic stability and present a critical contribution to our understanding of how DNA repair and replication intertwine.
In this work, using a genome wide CRISPR-Cas9 screen, SLX4IP loss was identified as synthetic lethal with loss of the Okazaki fragment maturation protein flap endonuclease 1 (FEN1). Synthetic lethality was due to defects in DNA replication, defined by an increase in single-stranded DNA (ssDNA) gap accumulation behind replication forks and elevated S-phase specific ADP-ribosylation, a hallmark for defects in Okazaki fragment maturation. SLX4IP is shown to target the SLX1-SLX4-XPF-ERCC1 nuclease complex to active replication forks upon loss of FEN1 to process lagging strand intermediates. SLX1 and XPF were further identified as the mediators of the lethality in FEN1 KO cells. Processing of lagging strand intermediates via SLX4IP in combination with SLX1-SLX4-XPF-ERCC1 provides an alternative mechanism to preserve lagging strand integrity. Together, these findings deliver novel insights into the contribution of structure-selective endonucleases and associated proteins to genomic stability and present a critical contribution to our understanding of how DNA repair and replication intertwine.
Version
Open Access
Date Issued
2024-09-28
Date Awarded
01/02/2025
License URL
Advisor
West, Stephen C.
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)