Adaptive use of error-prone DNA polymerases provides flexibility in genome replication during tumorigenesis.

Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics.

Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases.

An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.

Strategic targeting of Cas9 nickase induces large segmental duplications.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

NELF coordinates Pol II transcription termination and DNA replication initiation.

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD , a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTS: Expression of NELFCD transcript is up-regulated in colorectal tumors NELF-C protein is mandatory for the transition between G1-S phases during cell cycleNELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausingNELF-C loss leads Pol II to invade DNA replication initiation zones.