Award Title: A Novel Role for PARP Activity During Normal S phase and its Impact on Genome Stability and Cancer
Funder: Cancer Research UK

Abstract: Background:   The poly(ADP-ribose) polymerases (PARPs) comprise a superfamily of enzymes that modify themselves and other proteins with ADP-ribose. Three of these enzymes (PARP1, PARP2 and PARP3) are activated by DNA strand breaks including the archetypal family member, PARP1. Whilst numerous cellular roles have been identified for these enzymes in response to exogenous DNA damage/replication stress, their roles in normal unperturbed cells are unclear. PARP1 and PARP2 are of particular importance in this respect, because simultaneous deletion of both results in embryonic lethality in mouse, indicating that these two enzymes fulfil overlapping and essential role/s. Moreover, small molecule inhibitors of PARP1 are lethal in BRCA1/BRCA2-mutated cancer cells; a property now exploited therapeutically in the clinic. It is thus critical that we identify the roles of PARP activity during normal cell growth, to understand both how genome integrity and cellular viability are maintained and to further develop and exploit inhibitors of this activity in the clinic. To begin to identify the roles of PARP activity during normal cell growth we have identified the sites of endogenous poly(ADP-ribose) in chromatin. Surprisingly, we find that all of the endogenous poly(ADP-ribose) detectable in unperturbed wild-type human cells is localised not at sites of stochastic DNA damage but at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or inappropriate nucleotides incorporated during DNA replication nor does it result from DNA replication fork stress. Rather, based on exciting new data, we propose that the primary role of PARP activity during normal DNA metabolism is as a sensor of unligated Okazaki fragments during normal DNA replication, and as a facilitator of their subsequent repair.   Aims:   We will, 1. Characterise the novel role we have discovered for PARP activity during normal S phase  2. Address the importance of this role for cell survival and genome stability  3. Define how this role impacts on cancer incidence and treatment    Methods:  This Programme will employ molecular genetics, molecular biology, biochemistry, and cell biology.   How this research will be used:   This Programme will identify the role/s of PARP activity during normal S phase that are important for genome stability and cancer prevention, and identify the DNA structures that underpin the clinical sensitivity of specific types of cancer to PARP inhibitor. This work will also identify new cancer genetic backgrounds that are sensitive to PARP inhibitor, extending the clinical utility of this exciting class of anticancer drug.

Lay Abstract: Background:   The poly(ADP-ribose) polymerases (PARPs) comprise a superfamily of enzymes that modify themselves and other proteins with ADP-ribose. Three of these enzymes (PARP1, PARP2 and PARP3) are activated by DNA strand breaks including the archetypal family member, PARP1. Whilst numerous cellular roles have been identified for these enzymes in response to exogenous DNA damage/replication stress, their roles in normal unperturbed cells are unclear. PARP1 and PARP2 are of particular importance in this respect, because simultaneous deletion of both results in embryonic lethality in mouse, indicating that these two enzymes fulfil overlapping and essential role/s. Moreover, small molecule inhibitors of PARP1 are lethal in BRCA1/BRCA2-mutated cancer cells; a property now exploited therapeutically in the clinic. It is thus critical that we identify the roles of PARP activity during normal cell growth, to understand both how genome integrity and cellular viability are maintained and to further develop and exploit inhibitors of this activity in the clinic. To begin to identify the roles of PARP activity during normal cell growth we have identified the sites of endogenous poly(ADP-ribose) in chromatin. Surprisingly, we find that all of the endogenous poly(ADP-ribose) detectable in unperturbed wild-type human cells is localised not at sites of stochastic DNA damage but at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or inappropriate nucleotides incorporated during DNA replication nor does it result from DNA replication fork stress. Rather, based on exciting new data, we propose that the primary role of PARP activity during normal DNA metabolism is as a sensor of unligated Okazaki fragments during normal DNA replication, and as a facilitator of their subsequent repair.   Aims:   We will, 1. Characterise the novel role we have discovered for PARP activity during normal S phase  2. Address the importance of this role for cell survival and genome stability  3. Define how this role impacts on cancer incidence and treatment    Methods:  This Programme will employ molecular genetics, molecular biology, biochemistry, and cell biology.   How this research will be used:   This Programme will identify the role/s of PARP activity during normal S phase that are important for genome stability and cancer prevention, and identify the DNA structures that underpin the clinical sensitivity of specific types of cancer to PARP inhibitor. This work will also identify new cancer genetic backgrounds that are sensitive to PARP inhibitor, extending the clinical utility of this exciting class of anticancer drug.

Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health Categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE