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Function of poly(ADP-ribose) polymerase in response to DNA damage: gene-disruption study in mice
M Masutani1, T Nozaki, E Nishiyama
1Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan.
Abstract:
To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to gamma-irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following gamma-irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.
Insights
Poly(ADP-ribose) polymerase (PARP) plays a crucial role in DNA damage response. PARP deficiency increases sensitivity to DNA damage and influences cell-cycle arrest by regulating CDK activity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Poly(ADP-ribose) polymerase (PARP) is involved in DNA damage response.
- Understanding PARP's precise biological functions requires further investigation.
Purpose of the Study:
- To elucidate the biological functions of PARP in DNA damage responses.
- To investigate PARP's role in cell-cycle regulation.
Main Methods:
- Homologous recombination was used to disrupt the mouse PARP gene, creating PARP-deficient cell lines and mice.
- Genetic and biochemical assays were performed to assess PARP activity and cellular responses to DNA damage.
- Reconstituted reaction systems with purified PARP were used to study its effect on CDK activity.
Main Results:
- PARP-deficient cells exhibited increased sensitivity to gamma-irradiation and alkylating agents.
- PARP activity is not essential for p21 and Mdm-2 mRNA induction in the p53 pathway.
- Purified PARP suppressed pRB-phosphorylation activity, suggesting a role in CDK regulation and cell-cycle arrest.
Conclusions:
- PARP is physiologically important in the DNA damage response.
- PARP directly participates in the regulation of CDK activity, contributing to cell-cycle arrest.