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Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA
1Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
Abstract:
Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.
Insights
Human replication protein A (RPA) recognizes damaged DNA by sensing single-stranded character, not just double-stranded DNA. This binding, crucial for DNA repair, is mediated by RPA's single-stranded DNA-binding activity.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Protein-DNA Interactions
Background:
- Human replication protein A (RPA) is a vital heterotrimeric protein complex essential for cellular DNA metabolism.
- RPA is known to interact with damaged double-stranded DNA (dsDNA) and plays a role in nucleotide excision repair (NER).
Purpose of the Study:
- To investigate the mechanism by which RPA binds to both single-stranded DNA (ssDNA) and damaged dsDNA.
- To elucidate the specific DNA structural features and protein domains involved in RPA's interaction with damaged DNA.
Main Methods:
- Analysis of RPA binding affinities to ssDNA and dsDNA containing various types of DNA damage.
- Investigation of the role of RPA's ssDNA-binding activity and specific protein domains (zinc-finger domain) in DNA binding.
- Comparative affinity studies with other DNA repair proteins like XPA.
Main Results:
- RPA's affinity for damaged dsDNA is linked to helix disruption and requires its ssDNA-binding activity, indicating recognition of single-stranded character.
- RPA exhibits specific binding to damaged ssDNA, with a significant preference (up to 60-fold) for pyrimidine(6-4)photoproducts.
- Specific ssDNA binding is dependent on the zinc-finger domain in the 70-kDa RPA subunit, and RPA's affinity for damaged ssDNA is substantially higher than XPA's.
Conclusions:
- RPA recognizes damaged DNA by detecting localized single-stranded regions induced by DNA lesions.
- The zinc-finger domain of the 70-kDa subunit is critical for specific binding to damaged ssDNA.
- RPA likely interacts with both damaged and undamaged DNA strands within the NER complex, potentially facilitating efficient DNA excision and repair.