Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Apr 30;99(9):5848-53.
doi: 10.1073/pnas.082193799. Epub 2002 Apr 23.

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Karen M Vasquez  1 , Jesper Christensen, Lei Li, Rick A Finch, Peter M Glazer
Affiliations

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Karen M Vasquez et al. Proc Natl Acad Sci U S A. .

Abstract

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sequences of the APRT and supFG1 triplex target site duplexes and TFOs. (A) Nucleotide sequences of the APRT intron 1 target site duplex with the expected location and orientation of TFO binding. PTFO1 is the specific 19-base oligonucleotide that binds the APRT intron 1 site with high affinity. PTFOc is a 19-base control oligonucleotide that contains the same base composition as pTFO1, but a scrambled sequence and is not capable of binding the APRT intron 1 site. (B) The sequence of the modified APRT target site duplex, APRT-TA, containing a 5′ TA at the triplex-duplex junction for efficient psoralen crosslink formation. (C) The sequences of the supFG1 target site duplex and TFO. PAG30 binds the supFG1 site with high affinity, and the control oligonucleotide (pSCR30) does not. The psoralen intercalation site at the triplex-duplex junction is listed in bold for all duplex substrates. Psoralen is shaded and the base capable of monoadduct formation only. Lines indicate potential covalent interaction sites with psoralen after UVA irradiation.
Figure 2
Figure 2
Binding of human XPA and RPA proteins to psoralen-monoadducted triplex DNA. XPA (100 ng) and RPA (10 ng) proteins were coincubated with 196 bp DNA duplexes or triplexes containing the APRT intron 1 triplex target site at a concentration of 5 × 10−8 M. Protein–DNA interactions were assessed by electrophoretic mobility-shift assay (EMSA) on a 4% polyacrylamide gel buffered with TBE and 4% glycerol. Duplex sample lanes indicate the 196-bp DNA substrate alone, triplex + UV lanes indicate a 19-base psoralen-monoadducted triplex within the 196-bp duplex, control TFO lanes are samples containing the control oligonucleotide which does not form triplex, and HMT-duplex lanes indicate the 196-bp duplex treated with the free psoralen derivative (HMT) plus UVA irradiation in the absence of the TFO.
Figure 3
Figure 3
Concentration-dependent binding of RPA to covalent triplexes. RPA was incubated with the APRT 196-bp DNA substrate (5 × 10−8 M) at the concentrations indicated in the presence or absence of XPA (100 ng). The covalent triplex substrate indicates a psoralen-monoadducted triplex DNA structure. After substrate formation and incubation with XPA and RPA, the protein-DNA interactions were analyzed by EMSA. The HMT-duplex substrate is the 196-bp APRT substrate after incubation with HMT and UVA irradiation to induce psoralen photoadducts in the DNA duplex.
Figure 4
Figure 4
Recognition of a psoralen-crosslinked triplex by XPA and RPA. (A) The 196-bp modified APRT site (APRT-TA) containing a 5′ TA at the triplex-duplex junction was incubated with the specific psoralen-modified TFO, pTFO1, in the presence or absence of UVA irradiation to form a psoralen-crosslinked triple helix substrate (listed as triplex + UV) or a noncovalent triplex substrate (triplex − UV). Duplex APRT-TA also was incubated with the specificity control psoralen-modified oligonucleotide, pTFOc, (control TFO lanes) and substrates were incubated with RPA and XPA. The samples were subjected to EMSA analyses. All DNA substrates were at a concentration of 5 × 10−8 M. (B) The reactions were performed as in A, only the UVA irradiation dose was decreased to 0.18 J/cm2 so that crosslinked-triplex DNA substrate constituted less than half of the available DNA substrate in the triplex + UV lanes.
Figure 5
Figure 5
Binding of XPA and RPA proteins to short covalent triplex substrates. (A) The psoralen-modified TFOs were incubated with a radiolabeled 37-bp duplex substrate containing the APRT intron 1 site to form a triplex of 19 base triplets (5 × 10−8 M). Triplex + UV lanes indicate a psoralen-monoadducted triplex substrate incubated with XPA and RPA. Control TFO samples were incubated with the specificity control oligonucleotide (pTFOc). Samples were analyzed by EMSA after incubation with proteins. (B) The modified APRT site (APRT-TA) incorporated into a 37-bp synthetic duplex was incubated with pTFO1 to form a psoralen-crosslinked triplex substrate (triplex + UV lanes). Reactions with XPA and RPA were performed as described in A. Control TFO lanes indicate the 37-bp duplex incubated with the specificity control oligonucleotide, pTFOc that does not bind the triplex target site.
Figure 6
Figure 6
XPA and RPA recognize the supFG1 covalent triplex. A 188-bp plasmid fragment containing a 30-bp triplex target site in the supFG1 gene was incubated with a psoralen-modified TFO, pAG30, to form a triplex. Selected samples were irradiated with UVA (triplex + UV) as indicated to form psoralen-crosslinked triplex substrates (at 5 × 10−8 M) and then incubated with XPA and RPA as indicated by the (+) and (−) signs. The control TFO lanes contained radiolabeled duplex incubated with the control oligonucleotide, pSCR30. Protein binding was assessed by EMSA on a 4% polyacrylamide gel.
Figure 7
Figure 7
Antibody supershifting of XPA–RPA–DNA complexes and XPA-RPA binding interactions. (A) The psoralen-modified TFOs were incubated with a radiolabeled 57-bp duplex substrate to form a psoralen-crosslinked triplex of 30 base triplets at a concentration of 5 × 10−8 M. Triplex + UV lanes indicate a psoralen-crosslinked 57-bp supFG1 triplex substrate incubated with XPA and RPA proteins. Antibodies against XPA (XPA ab) or RPA (RPA ab) were added to the indicated lanes. Duplex lanes contain the 57-bp duplex only. Samples were analyzed by EMSA after incubation with proteins and subjected to autoradiography. (B) Control samples were treated as described above with proteins and antibodies incubated with the psoralen-crosslinked 57-bp triplex substrate only. (C) ELISA analysis of XPA and RPA binding. Purified recombinant proteins [RPA, proliferating cell nuclear antigen (PCNA), or BSA] were coated on 96-well plates and then incubated with increasing amounts of XPA protein. Specific binding interactions were detected as OD units after incubation with anti-XPA primary antibody and a horseradish peroxidase-conjugated secondary antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Auerbach A D, Verlander P C. Curr Opin Pediatr. 1997;9:600–616. - PubMed
    1. Berneburg M, Lehmann A R. Adv Genet. 2001;43:71–102. - PubMed
    1. Cleaver J E, Thompson L H, Richardson A S, States J C. Hum Mutat. 1999;14:9–22. - PubMed
    1. de Boer J, Hoeijmakers J H. Carcinogenesis. 2000;21:453–460. - PubMed
    1. Woods C G. Arch Dis Child. 1998;78:178–184. - PMC - PubMed

Publication types

LinkOut - more resources

-