RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

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.

Astragalus polysaccharide protects formaldehyde-induced toxicity by promoting NER pathway in bone marrow mesenchymal stem cells

“…RPA can protect single-stranded DNA from nuclear degradation and hairpin formation after DNA damage [43]. At the same time, RPA1 and RPA2 can bind to XPA to form the RPA-XPA complex, which is involved in the recognition of DNA damage [44]. In this study, we found that the mRNA and protein expression of RPA1, RPA2 decreased in FA-treated cells compared with control cells, indicating that RPA1, RPA2 did not participate in the repair of DNA damage caused by FA.…”

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair

“…The structure containing a damaged flap and a 26 nt gap imitates the DNA intermediate arising in the NER process after damaged strand cleavage by XPF‐ERCC1; DNA with the same flap and a 10 nt gap can be attributed to the intermediate of the subsequent partial gap filling . Crosslinking with 5‐I‐dUMP containing flap‐gap26 DNA revealed that RPA interacts with the ssDNA track in the gap and does not utilize the flap . Based on this study, it may be supposed that, after XPF‐ERCC1 incision, RPA remains bound with the undamaged strand in the gap created and can be translocated on the flap when gap is shortened during the resynthesis step (Fig.…”

“…From the NER factors, only RPA participates during all stages of the repair process. Using 60‐mer DNA duplexes containing a 31 nt flap with the fluorescein residue mimicking the bulky lesion and a 26 or 10 nt gap, RPA and XPA interactions with intermediates of the NER late stages were analyzed . The structure containing a damaged flap and a 26 nt gap imitates the DNA intermediate arising in the NER process after damaged strand cleavage by XPF‐ERCC1; DNA with the same flap and a 10 nt gap can be attributed to the intermediate of the subsequent partial gap filling .…”

Interface of DNA Repair and Metabolism

“…The NER operates through two subpathways: a global genome repair (GG-NER) and transcription coupled repair (TC-NER). The GG-NER subpathway acts on lesions present in the whole genome, with XPC and XPE as the main proteins that recognize the DNA damage, while the TC-NER recognizes lesions during transcription elongation, when the RNA polymerase II is stalled by DNA damage found in the template strand [101,102].…”