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Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1
M K Shivji1, E Ferrari, K Ball
1Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Abstract:
The p21Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the approximately 30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could also inhibit filling of model 30 nt gaps by both purified DNA polymerases delta and epsilon. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.
Insights
The protein p21Cdn1 (cip1/waf1/sdi1) inhibits DNA repair synthesis by binding to PCNA, but this effect is transient in cell extracts. This allows DNA repair to proceed efficiently.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- p21Cdn1 (cip1/waf1/sdi1) protein inhibits cell proliferation by targeting cyclin-Cdk complexes.
- p21 also inhibits DNA replication by binding to PCNA, a key factor in DNA synthesis.
- DNA nucleotide excision repair (NER) is PCNA-dependent but continues despite p21 induction in damaged cells.
Purpose of the Study:
- Investigate the mechanisms of p21-resistant DNA repair, specifically NER.
- Determine how p21 interacts with PCNA during the NER pathway.
- Characterize the inhibitory effects of p21 on DNA repair synthesis in human cell extracts.
Main Methods:
- Utilized a direct end-labeling assay to quantify NER excision of damaged oligonucleotides.
- Assessed the impact of p21-PCNA binding on DNA gap filling by purified polymerases and cell extracts.
- Employed a synthetic p21 peptide mimicking PCNA binding to evaluate inhibition of NER synthesis.
Main Results:
- p21 did not inhibit the excision step of NER.
- Pre-binding p21 to PCNA inhibited gap filling by DNA polymerases delta and epsilon, particularly when uncoupled from incision.
- Inhibition of repair synthesis by p21 in cell extracts was reversible over time, suggesting dissociation from PCNA.
- p21 blocks polymerase association with PCNA but not PCNA loading onto DNA, enabling rapid repair synthesis upon p21 dissociation.
Conclusions:
- p21's inhibition of NER gap filling is dependent on its interaction with PCNA.
- The transient nature of p21-PCNA interaction in cell extracts allows for efficient DNA repair synthesis.
- A synthetic p21 peptide effectively inhibits NER synthesis, highlighting the importance of the p21-PCNA interaction in this process.