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.

Coordination of Primer Initiation Within the Catalytic Domain of Human PrimPol.

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.

Primase-polymerases: how to make a primer from scratch.

To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.

Molecular basis for the initiation of DNA primer synthesis.

During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle.

Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, we demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol's RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle.

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication.

To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.