Cellular Responses to Widespread DNA Replication Stress

Abstract

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

Keywords: DNA damage; DNA damage response; DNA double-strand breaks; genome instability; oxidative DNA damage; replication stress; structure-specific nucleases.

Figure 1

Nucleic acid structures that cause replication stress. These include difficult-to-replicate sequences such as homopolymeric nucleotide runs, palindromes and triplet repeats that can form stem-loop or cruciform structures, G-quadruplex DNA, and self-invading loops at telomeres. Stable R-loops cause replication stress when encountered by replicative DNA polymerases.

Figure 2

Lesion bypass mechanisms. (A) TLS polymerases can synthesize across DNA lesions (red symbol), but with increased mutagenesis. (B) Repriming past DNA lesions is accurate but results in SS gaps (dashed line). (C) Template switching is an accurate lesion bypass mechanism that involves strand invasion of the sister chromatid for accurate lesion bypass. (D) Blocked replication forks can be rescued by an adjacent fork, but similar to repriming, replication is incomplete as it leaves an SS gap.

Figure 3

Replication fork reversal, protection, and restart mechanisms. Blocked forks are reversed to a 4-way branched structure (chicken foot) that presents a seDSB. This allows the blocked polymerase to be extended using the nascent sister strand as template (dashed line). Limited fork reversal driven by indicated fork remodeling proteins can be regressed by RECQ1-mediated branch migration to restart the fork. Alternatively, more extensive reversal generates a longer strand at the single-end DSB that is resected and bound by fork protection factors to prevent degradation of the seDSB end by the indicated nucleases.

Figure 4

Blocked forks are cleaved by MUS81-EME2 or EEPD1, creating seDSBs. Resection creates ssDNA that is bound by RAD51 to catalyze strand invasion for HR-repair of the broken replication fork. Cleavage by MUS81-EME2 may require extra time for Okazaki fragment maturation before strand invasion.

Figure 5

TATDN2 RNase promotes survival of BRCA1-defective cells by suppressing R-loop-induced replication stress. (A) TATDN2 is a structure-specific RNase that degrades RNA in R-loops with both exonuclease and endonuclease activities. (B) BRCA1 helps cells manage R-loops to prevent toxic replication stress. With functional BRCA1, adding or deleting miR-4638-5p or TATDN2 does not affect cell viability. In BRCA1-deficient cells, IRE1 RNase levels increase, reducing miR-4638-5p and increasing TATDN2 which acts to limit R-loops and associated replication stress, thereby promoting cell viability. Expressing miR-4638-5p or downregulating TATDN2 kills BRCA1-deficient cells due to increased R-loop-associated replication stress.