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Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of
L A Henricksen1, T Carter, A Dutta
1Department of Biochemistry, University of Iowa, Iowa City, 52242, USA.
Abstract:
The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.
Insights
Replication Protein A (RPA) phosphorylation by DNA-PK is regulated by RPA32's N-terminus. This N-terminus is crucial for overcoming DNA-PK's inhibitory effects on DNA replication, suggesting indirect modulation of replication.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Replication
Background:
- Replication Protein A (RPA) is a vital heterotrimeric complex for DNA metabolism.
- The 32 kDa subunit (RPA32) of RPA is a target for cellular kinases like DNA-dependent protein kinase (DNA-PK).
Purpose of the Study:
- To investigate the role of RPA32's N-terminus in RPA phosphorylation and its effect on DNA replication.
- To determine if DNA-PK phosphorylation of RPA directly impacts DNA replication.
Main Methods:
- Purification of a mutant human RPA (rhRPA) lacking the first 33 amino acids of RPA32 (rhRPA x 32delta1-33).
- In vitro phosphorylation assays using DNA-PK and wild-type or mutant RPA.
- SV40 DNA replication assays to assess the functional impact of RPA phosphorylation.
Main Results:
- The N-terminal residues 1-33 of RPA32 contain all DNA-PK phosphorylation sites.
- The mutant rhRPA x 32delta1-33, lacking these N-terminal residues, was not phosphorylated by DNA-PK.
- While rhRPA x 32delta1-33 supported DNA replication, its pre-treatment with DNA-PK strongly inhibited replication, unlike wild-type RPA.
Conclusions:
- The N-terminus of RPA32 is essential for mediating RPA's interaction with DNA-PK and preventing replication inhibition.
- RPA phosphorylation by DNA-PK likely modulates DNA replication indirectly by affecting interactions with other replication factors.