Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG

The XPA Protein—Life under Precise Control

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA’s scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein’s activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein

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

<div><p>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.</p></div

RPA-XPA complex formation. Fluorescently labeled RPA was titrated by XPA in the absence of DNA or in the presence of unlabeled (A) or ³²P-labeled (B) ssDNA.

<p>The reaction mixtures (20 μl) contained buffer A, 10 nM Flu-RPA and indicated concentrations of XPA and/or DNA. A schematic view of the DNA structures is presented at the top: the triangle indicates the position of the bulky lesion. (<b>A</b>) The right panel shows the quantitative analysis of the data from the EMSA experiments. Percentage of the XPA protein active in DNA binding was 20%. Averages and standard deviations were estimated from three independent experiments.</p

Structures of model DNA used in the study.

<p>For oligonucleotide sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190782#pone.0190782.s001" target="_blank">S1 Table</a>.</p

Comparative analysis of RPA and XPA binding to DNA containing a 10 nt gap with a flap (A) or a 10 nt gap only (B).

<p>The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-³²P-labeled DNA and the indicated concentrations of proteins. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. White circles indicate putative RPA-XPA-DNA complexes with unknown stoichiometry.</p

Comparative analysis of RPA and XPA binding to DNA containing the 26 nt gap with the flap (A) or the 26 nt gap only (B).

<p>The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-³²P-labeled DNA and the indicated concentrations of proteins. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. White circles indicate putative RPA-XPA-DNA complexes with unknown stoichiometry.</p

XPA binding to DNA containing a 10 or 26 nt gap (A) or both a gap and a flap (B). (C) XPA binds bubble-DNA more effectively than flap-gap containing DNA.

<p>Averages and standard deviations were estimated from three independent experiments.</p

RPA binding to DNA containing a flap with a gap (A, B) or a gap only (C).

<p>The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-³²P-labeled DNA and RPA at the indicated concentrations. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. (<b>A</b>) Increasing amounts of RPA were added to DNA containing a 26 nt gap and a flap. (<b>B</b>) RPA was added to DNA containing a 10 nt gap and a flap. (<b>C</b>) RPA was added to DNA containing only gaps: a 10 nt gap (lanes 1–11) or a 26 nt gap (lanes 12–22). (<b>D</b>) Summary plot of the data of the binding experiments shown in panels <b>A</b>-<b>C</b>. Averages and standard deviations were estimated from three independent experiments.</p