Nucleases and Co-Factors in DNA Replication Stress Responses

Abstract

DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics.

Cellular Responses to Widespread DNA Replication Stress

“…This corresponds to the large increase in genome size and complexity [187], which likely increased intrinsic replication stress. As we discussed previously [188], 3 fork cleavage by MUS81 generates an end that once resected and coated with RAD51 must invade the lagging strand duplex, whereas 5 cleavage by EEPD1 generates an end that can invade the leading strand duplex (Figure 4). Because the lagging strand comprises discontinuous Okazaki fragments near the replication fork, strand invasion into the lagging strand may be less efficient or perhaps delayed until Okazaki fragment processing is completed.…”

POLQ mediates replication-stress induced structural variant formation throughout common fragile sites during mitosis

“…Both the MUS81 and GEN1 structure-specific nucleases are required for CFS expression 77, 78 and are excellent candidates for creating those source DSBs leading to SV formation. If our slippage model is correct, it implicates MUS81 as the primary nuclease as it is thought to cleave the leading strand template at stalled forks, the orientation that would yield 5’ overhangs 79 . Such symmetrical cleavage of stalled forks has been proposed to lead to HR-independent SCEs 80 where DSB repair by TMEJ would obligatorily lead to SV formation, especially deletions corresponding to unreplicated DNA spans.…”

Targeting Replication Stress Response Pathways to Enhance Genotoxic Chemo- and Radiotherapy

“…There is significant crosstalk among the PIKKs, as certain autophosphorylation targets may be phosphorylated by other PIKKs under specific conditions [ 40 , 41 , 42 ]. Replication protein A (RPA) has several residues targeted by multiple PIKKs, demonstrating further crosstalk [ 43 , 44 ]. Despite this crosstalk, the primary roles of DNA-PKcs and ATM are to promote the repair of two-ended (frank) DSBs by cNHEJ and HR, respectively ( Figure 1 A).…”

“…In contrast, the 5′ nuclease EEPD1 evolved more recently in chordates/vertebrates, and this mode of cleavage allows invasion by the resected end into the continuous, leading strand duplex. The EEPD1 cleavage mechanism may speed fork restart and thereby reduce the opportunity for stressed forks to assume toxic structures, promoting both genome stability and cell survival under stress [ 43 ].…”

“…Several nucleases play key roles in the replication stress response [ 43 ]. CtIP is a nuclease and key factor in the early steps of DNA end resection at DSBs, functioning with BRCA1 in both end resection and the protection of stressed replication forks [ 171 , 172 , 173 ].…”