Abstract
Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.
Highlights
Organism survival depends on efficiently maintaining the integrity of the genome, such as accurate replication of base pairs as well as identifying and repairing a variety of DNA lesions
xeroderma pigmentosum group A (XPA) and Replication protein A (RPA) form stable complexes that are not disrupted in the presence of DNA. Both RPA and XPA are indispensable Nucleotide excision repair (NER) factors required for repair process in vivo and in vitro [15, 50]
We found that XPA binds to DNA mimicking post-incision intermediates with less affinity than to bubble-DNA intermediate (Fig 4C) whereas RPA has some preference for DNA mimicking NER intermediates formed after the action of XPF-ERCC1
Summary
Organism survival depends on efficiently maintaining the integrity of the genome, such as accurate replication of base pairs as well as identifying and repairing a variety of DNA lesions. Nucleotide excision repair (NER) is the only mechanism in humans that can repair double helix-distorting lesions, such as UV-induced pyrimidine dimers or bulky chemical adducts caused by environmental carcinogens or chemotherapeutic agents. NER operates through two distinct pathways: global genome NER (GG-NER) and transcription coupled NER (TC-NER), where the lesion-stalled RNA polymerase II triggers the assembly of the repair machinery at the site of damage [1, 2]. The minimal set of proteins required to perform complete GG-NER (more than 30 polypeptides) was established using an in vitro reconstituted system [3].
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