Investigating the Mechanisms Controlling Homologous Recombination-Dependent DNA Replication Fork Recovery in Response to Replication Stress.

Before cells divide, DNA replication duplicates the genome, so that a copy of all chromosomes may transmitted to each of two incipient daughter cells. To achieve this, the parental chromosomes become unwound at origins of replication, forming DNA replication forks. These are the sites where replicative DNA polymerases catalyse DNA synthesis. As replication forks track along the chromosomes, they are routinely stalled by a range of obstacles including polymerase-blocking DNA lesions, DNA secondary structures, DNA-binding proteins, and DNA-RNA hybrids; this is referred to as DNA replication stress. Unfavourable replication conditions, such as those found in dysregulated cancer cells, further enhance replication stress. As a consequence, replication forks stall or break, jeopardising genome duplication, and exposing cells to chromosome segregation problems, chromosome breakage, and gross chromosomal instability. To offset these threats to genome stability, cells have evolved replication fork recovery mechanisms. Notably, perturbed fork recovery has been linked to human diseases including primordial dwarfism and tumorigenesis. Conversely, because cancer cells generate intrinsic replication stress, targeting replication fork recovery pathways has emerged as a potential anti-cancer strategy. Thus, understanding the mechanisms and regulation of replication fork recovery is of great scientific interest and biomedical importance.
One of the ways in which cells reboot DNA replication at stalled replication forks is dependent upon homologous recombination. This requires the dissociation of a nascent DNA strand, which undergoes invasion of the parental chromosome to from a so-called displacement loop (D-loop). D-loop DNA synthesis, which uniquely depends upon the polymerase subunit POL32 (POLD3), then becomes the new mode of replication. This process is generally beneficial, helping cells to overcome even tenacious replication obstacles. However, D-loop DNA synthesis is error prone and unstable, which can cause ectopic recombination and chromosome rearrangements. A key question, therefore, is how cells control recombination-dependent replication fork recovery to balance the benefits to replication completion with the risks the pathway poses to genome stability.
We have recently reported that the disease-associated DNA2 nuclease/helicase is a critical processing factor at stalled replication forks, strictly required for the completion of chromosome replication. Furthermore, we suggested that the actions of DNA2 limit the use of recombination-dependent fork restart, and that excessive recombination in the absence of DNA2 is toxic for cells. This recombination "gatekeeper" model has provided a new rationale for the essential nature of DNA2 across organisms, but remains to be tested. Consistent with this model, we have identified new Pol32 separation-of-function mutations that specifically disable D-loop DNA synthesis, and concomitantly rescue the viability of DNA2-defective cells. Here, we propose to exploit these findings to unlock key questions of how cells control homologous recombination at stalled replication forks and implement the appropriate balance of recovery pathways in the restart of DNA replication. We will test the DNA2 gatekeeper hypothesis directly at a defined genomic site of replication stalling. Secondly, we will leverage the interactions between DNA2 and POL32 to reveal the elusive requirement of POL32 for D-loop DNA synthesis. And thirdly, we will examine how cells instruct Pol32 by post-translational modifications we identified, to act as a rheostat controlling the levels of recombination-mediated replication.
This work programme will provide unprecedented mechanistic insight into the roles of DNA2 and POL32 (POLD3) in controlling replication fork recovery and offsetting replication stress. The conclusions will help rationalize DNA2 and POLD3 disease links with Seckel syndrome, mitochondrial myopathy, and cancer.

DNA replication stress (RS) ensues when replication fork (RF) progression is perturbed by physical obstruction or oncogene-induced cell-cycle dysregulation. It is increasingly evident that RS, a hallmark of cancer, drives genome instability. There is intense interest in the mechanisms that cells employ to recover stalled and broken RFs.
Recently, we demonstrated that the disease-associated DNA2 nuclease/helicase is required for stalled RF recovery and the completion of chromosome replication. Moreover, we suggested that DNA2 dysfunction results in excessive homologous recombination (HR) at stalled RFs, entailing DNA synthesis in the context of displacement loops (D-loops), mediated by Pol delta subunit POL32 (POLD3) through an unknown mechanism. We hypothesized that promiscuous HR-dependent replication, by way of D-loop collapse, exposes checkpoint-activating single-stranded DNA. Thus, cells require DNA2 to limit HR-dependent RF recovery, or die from DNA damage checkpoint hyperactivation.
Here, we will test the hypothesis that DNA2 functions as a gatekeeper to HR-mediated RF restart by determining the replication dynamics at a site-specific RF barrier. Leveraging genetic interactions between DNA2 and newly identified separation-of-function mutants of POL32, which specifically disable D-loop DNA synthesis, we will unlock the enigmatic biological mechanism that makes POL32 (POLD3) indispensable for HR-dependent replication. Finally, we will pursue preliminary results of Pol32 post-translational modifications to elucidate how the protein, rather than being a mere facilitator of D-loop DNA synthesis, acts as a rheostat controlling the extent of HR-mediated replication.
We expect our findings to reveal unprecedented mechanistic insight into how cells regulate HR-dependent RF recovery, and to help elucidate disease links of DNA2/POLD3 with Seckel syndrome, mitochondrial myopathy, and cancer. The work will inform efforts of developing DNA2 into an anti-cancer target.