Nascent DNA Strand Maturation in Human Cells and its Targeting by Inhibitors of Poly(ADP-Ribose) Metabolism

Background: Inhibitors of the DNA strand break sensor protein poly(ADP-ribose) polymerase-1 (PARP1) are now employed in the clinic, to treat cancers with hereditary mutations in BRCA1 and BRCA2.

In our recent work, we have discovered that the primary source of PARP1 activity in normal proliferating cells are partially processed nascent strand intermediates of DNA replication known as Okazaki fragments.

We have established that a major consequence of treating cells with PARP1 inhibitors is the blocked maturation of nascent strands during DNA replication, and that there exists in human cells a temporal and spatial interplay between the different nascent strand maturation pathways.

In particular, whereas the canonical Okazaki fragment processing machinery is primarily responsible for the maturation of small nascent strand fragments located close to DNA replication forks, the PARP-dependent pathway is primarily responsible for the maturation of large nascent strand fragments that have escaped the canonical pathway and are located far behind replication forks.

In addition, excitingly, we have now also discovered a requirement for homologous recombination (HR)-mediated repair for the maturation of large nascent strand intermediates, if PARP-dependent processing is blocked by PARP inhibitor.

We propose that this HR-mediated process, which requires the tumour suppressor BRCA2, reflects the post-replication repair of single-strand gaps present in nascent strands; an enigmatic process reported in bacteria and yeast but which in human cells is poorly understood.

Finally, we have also discovered that defects in canonical Okazaki fragment processing confer exquisite sensitivity to inhibitors of both PARP1 and PARG, highlighting a general vulnerability to inhibitors of poly(ADP-ribose) metabolism that we propose can be exploited for cancer therapy.

Aims: In the current application, we will test the model described above and uncover the molecular mechanisms by which human cells ensure the integrity of nascent strands during DNA replication; a process critical for genome stability and tumour prevention.

In addition, we will test our hypothesis that defects in canonical Okazaki fragment maturation confer a vulnerability to inhibitors of poly(ADP-ribose) metabolism that can be exploited in the cancer clinic.