Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place

Abstract

Oncogene activation disturbs cellular processes and accommodates a complex landscape of changes in the genome that contribute to genomic instability, which accelerates mutation rates and promotes tumorigenesis. Part of this cellular turmoil involves deregulation of physiologic DNA replication, widely described as replication stress. Oncogene-induced replication stress is an early driver of genomic instability and is attributed to a plethora of factors, most notably aberrant origin firing, replication-transcription collisions, reactive oxygen species, and defective nucleotide metabolism.Significance: Replication stress is a fundamental step and an early driver of tumorigenesis and has been associated with many activated oncogenes. Deciphering the mechanisms that contribute to the replication stress response may provide new avenues for targeted cancer treatment. In this review, we discuss the latest findings on the DNA replication stress response and examine the various mechanisms through which activated oncogenes induce replication stress. Cancer Discov; 8(5); 537-55. ©2018 AACR.

Figure 1

DNA replication and replication stress response. A, in late mitosis and throughout G1-phase, the pre-replicative complex comprised by the ORC complex, CDC6 and CDT1 is recruited to replication origins to facilitate loading of the MCM2-7 complex. During G1-phase, retinoblastoma (Rb) is bound to E2F rendering it inactive. Phosphorylation of Rb by the Cyclin D-CDK4 complex alleviates its inhibitory effect on E2F. APC/C activity is high from late M- to late G1-phase regulating CDK activity. Upon entry in S-phase, APC/C is inhibited and CDKs are activated throughout S, G2 and early M-phase. CDKs form complexes with E2F-regulated cyclins that collaborate with CDC7 to phosphorylate TRESLIN and MCM2-7 complex, activating the CMG (CDC45-MCM2-7-GINS) helicase complex. Simultaneously, clamp loader RFC and sliding clamp PCNA are recruited and enable polymerases δ and ε to initiate replication in the lagging and leading strands respectively. B, when a replication fork is stalled, ssDNA is generated as the CMG complex unwinds DNA. ssDNA binds RPA that recruits ATR (through ATRIP), RAD17-RFC and 9-1-1 complexes at the stalled fork. ATR is then activated by TOPBP1/RAD17/9-1-1 and ETAA1 and phosphorylates H2AX, while through TIMELESS/TIPIN/CLASPIN phoshoprylates CHK1. CHK1 then organises the RS response by arresting the cell cycle, inhibiting new origin firing, enabling dormant origin firing and stabilizing the fork, which can then be reversed by various proteins in a chicken foot structure. An unprotected reversed fork is susceptible to nucleolytic degradation by MRE11, EXO1 and DNA2. A stalled fork can restart through homologous recombination, re-priming, template switching, translesion synthesis or break-induced replication. Alternatively, it will collapse into DSBs by the combined activity of MUS81-EME1, XPF-ERCC1, EXO1 and SLX4 that will drive the cell to senescence.

Figure 2

Repair of a stalled fork. Upon encountering an obstacle, replication machine arrests and the CMG complex unwinds DNA ahead of the stalled fork, leaving ssDNA behind. Replication can resume through various mechanisms, such as translesion synthesis, template switching, re-priming, break-induced replication or homologous recombination. If this fails, the stalled fork is cleaved by MUS81-EME1 during G2-phase and upon entrance in metaphase RAD52, POLD3 and MUS81-SLX4 collaborate to facilitate replication of the under-replicated DNA through MiDAS. In the absence of MiDAS and during chromosomal segregation in anaphase, UFBs are formed at the CFSs, which will appear as micronuclei or 53BP1 bodies in the daughter cells.

Figure 3

Oncogene-induced genomic instability. A, germline mutations in DNA repair genes lead to GIN. Activated oncogenes elicit genomic instability by causing defects in DNA repair, telomere erosion or replication stress; see text for details. B, activated oncogenes induce a multifaceted set of intertwined activities that deregulate fork progression leading to under-replicated DNA and genomic instability. In particular, through deregulation of the Rb/E2F pathway licensing factors are increased which instigates origin re-firing that decreases fork speed in response to head-to-tail fork collisions. Deregulation of CDK activity can decrease or increase origin firing. In the first case, fork speed is initially increased, but the cell ends up with under-replicated DNA due to its inability to rescue endogenous RS by firing dormant origins. Increased origin firing raises the possibility of TRCs and simultaneously may cause depletion of dNTPs, histones or RPA. Oncogene-induced ROS either increase origin firing or oxidize nucleotides that potentially may affect fork progression. Oncogenes also increase transcription activity that either directly or through R-loops enhance TRCs.

Figure 4

Transcription-associated replication stress. A, upon head-on conflicts between replication and transcription, R-loop formation is increased and ATR/CHK1 are activated. B, upon co-directional conflicts between replication and a stalled or backtracked RNAP, DSBs are formed that activate ATM/CHK2 and R-loops are resolved. C, in response to any obstacle an RNAP may pause and backtrack and act as an impediment to fork progression; D, this can also occur as a result of an encounter with an R-loop. In both cases, this will cause accumulation of arrested or backtracked RNAPs that hinder fork progression. E, while the replication and transcription machineries move, positive supercoiling develops in front of them, that can impede progression of both. F, transcribed RNA may get trapped in the nuclear pore complex generating obstacles to fork progression. G, R-loops can cause chromatin condensation, marked by H3 phosphorylation at Ser-10, that impedes fork progression. H, G-quadruplexes can be formed co-transcriptionally or within the ssDNA part of an R-loop and act as obstacles to fork progression.

Figure 5

Nucleotide and ROS metabolism as a cause of replication stress. dNTPs are formed either through the de novo pathway from glutamine, glycine, folic acid, aspartate, 5-phosphoribosyl-1-pyrophosphate or by nucleoside degradation through the salvage pathway. The reduction of ribonucleosides to dNTPs is catalysed by the ribonucleotide reductase (RNR), which is compromised by two catalytic (RRM1) and two regulatory (RRM2, RRM2B) subunits. Oncogenes may target RNR activity or induce hyper-proliferation that will both reduce dNTP pool affecting fork progression. Oncogenes also induce production of ROS, including O₂^ˉ, H₂O₂ and OH•. O₂^ˉ is produced either by oxidation of NADPH by NOX enzymes or through aerobic respiration in mitochondria. H₂O₂ is generated by O₂^ˉ which is converted by superoxide dismutase (SOD), while OH• are produced from H₂O₂ in the presence of Fe⁺². It is not clear if oxidized dNTPs affect replication fork speed.