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
. 2009 Jul;37(13):4420-9.
doi: 10.1093/nar/gkp399. Epub 2009 May 25.

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

Junhua Zhao  1 , Aklank Jain, Ravi R Iyer, Paul L Modrich, Karen M Vasquez
Affiliations

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

Junhua Zhao et al. Nucleic Acids Res. 2009 Jul.

Abstract

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA-RPA and XPC-RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta interacted with XPA-RPA or XPC-RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Specific recognition of Tdp-ICLs by MutSβ. (A) Sequences of the 57-bp DNA duplex (complementary oligonucleotides 71 + 72) and the 5′-psoralen-conjugated TFO (pAG30) used to form the Tdp-ICL substrate. The 5′-TpA psoralen crosslinking site is shown in the box. (B) EMSA analysis of MutSβ (43 nM) on Tdp-ICLs (10 nM) with unlabeled Tdp-ICLs or unlabeled DNA duplex used as competitor. The concentrations of the competitors were at 2×, 10× and 50× of the 5′ end γ-32P labeled Tdp-ICL substrate. Purified MutSβ was incubated with labeled Tdp-ICLs at 30°C for 5 min prior to the addition of the competitor DNA. Incubation was then continued for 20 min. The resulting DNA–protein complexes were separated from the free DNA substrate and unbound protein using 6% native PAGE. The gel was then dried and scanned using a PhosphorImager. Lane 1, DNA substrate only; lane 2, MutSβ; lanes 3–5, MutSβ with additional unlabeled competitor Tdp-ICL substrate; lanes 6–8, MutSβ with additional unlabeled competitor duplex DNA. (C) Quantitation of Mutsβ bound to the Tdp-ICL (radioactivity in the band containing MutSβ as a percentage of the total radioactivity loaded in the lanes from Figure 1B). (D) Southwestern blotting analysis of the MutSβ–DNA complex and unbound MutSβ. The MutSβ–DNA complex or MutSβ alone was separated using 4% native PAGE, and then transferred onto a PVDF membrane after exposure to X-ray film. The membrane was probed with an α-MSH2 antibody to detect MutSβ.
Figure 2.
Figure 2.
MSH2 binds to a Tdp-ICL in human cells. (A) Schematic representation of plasmid, pSupFG1, containing the same TFO binding site as in the synthetic duplex 71 + 72, adjacent to a psoralen crosslinking site, and primer sites (P1, P2, P3 and P4) on the plasmid used for ChIP analysis. (B and C) Representative agarose gels of PCR products from ChIP assays demonstrating the binding of the α-MSH2 or α-MSH3 antibody to DNA near the site of the Tdp-ICL. Purified DNA was analyzed by standard PCR methods using primers P1 and P2 (near the TFO binding and psoralen crosslinking site) and control primers P3 and P4 (∼2 kb from the TFO binding and psoralen crosslinking site). Lane 1, PCR control without input template; lanes 2–7, PCR products amplified with primers P1 & P2; lanes 8–13, PCR products amplified with primers P3 & P4; C, PCR control without template; ‘–’ template from untransfected cells; ‘+’ template from the cells transfected with plasmid; G, pulldowns with α-IgG antibody; P, template from the cells transfected with the control plasmid; X, template from the cells transfected with the plasmid containing the Tdp-ICL. M, 100 bp DNA ladder (Bio-Rad).
Figure 3.
Figure 3.
Formation of complexes of MutSβ and XPA–RPA on Tdp-ICLs. (A) The purified human recombinant protein complexes MutSβ (43 nM) and XPA–RPA (XPA at 60 nM; RPA at 4 nM, pre-incubated) were added alone or together to Tdp-ICLs (10 nM). The protein–DNA complexes are indicated by arrows. An unlabeled Tdp-ICL or DNA duplex competitor was added to the reaction at 2×, 10× and 50× the [γ-32P]dATP-labeled Tdp-ICL substrate concentration (10 nM). Lane 1, DNA substrate only; lanes 2–4, DNA substrate with indicated proteins; lanes 5–7, MutSβ and XPA–RPA with increased unlabeled Tdp-ICL competitor; lanes 8–10, MutSβ and XPA–RPA with increased unlabeled DNA duplex competitor. Binding of multiple MutSβ molecules to Tdp-ICLs is indicated with a black dot. (B) Quantitation of MutSβ and XPA–RPA bound to the Tdp-ICL (radioactivity in the band containing both MutSβ and XPA–RPA as a percentage of the total radioactivity loaded in the lanes from Figure 3A). (C) Antibody supershift assay of MutSβ and XPA–RPA on the Tdp-ICL. The purified protein complexes were incubated with the Tdp-ICLs at 30°C for 5 min and then incubated with an antibody (α-RPA/p34 or α-MSH3, 0.2 μg each) for 20 min. The DNA–protein complexes were separated from the free DNA substrate using 6% native PAGE. The protein–DNA complexes are indicated by arrows. The triangle marks the XPA–RPA–Tdp-ICL complex shifted by an α-RPA/p34 antibody; the double triangle marks the MutSβ–XPA–RPA–Tdp-ICL complex shifted by an α-RPA/p34 antibody; the star marks the MutSβ–Tdp-ICL complex shifted by an α-MSH3 antibody; the double star marks the MutSβ–XPA–RPA–Tdp-ICL complex shifted by an α-MSH3 antibody.
Figure 4.
Figure 4.
MutSβ and XPA–RPA interact on Tdp-ICLs. (A) EMSA analysis of purified MutSβ and XPA–RPA complexes with decreasing concentrations of the Tdp-ICL substrates. Lane 1, DNA substrate; lane 2, MutSβ; lane 3, XPA–RPA and MutSβ; lanes 4–6, MutSβ and XPA–RPA with decreasing amounts of DNA substrate (1/2×, 1/4× and 1/8×, respectively, of 10 nM). The protein–DNA complexes are indicated by arrows. (B) Quantitation of MutSβ–XPA–RPA–Tdp-ICL complex bands and duplex DNA bands as the percentage of total DNA substrate (Sub) loaded in lanes 3–6 in Figure 4A. (C) EMSA analysis of the Tdp-ICLs with sequential addition of purified MutSβ or XPA–RPA. Lane 1, DNA substrate; lane 2, MutSβ; lane 3; XPA and RPA; lane 4, MutSβ and XPA–RPA; lanes 5–7, samples were incubated with MutSβ at 30°C for 10 min, then XPA–RPA (at 1×, 2× and 5× of 60 nM/4 nM) was added, and the samples were incubated for another 10 min. Lanes 8–10, samples were incubated with XPA–RPA at 30°C for 10 min, then MutSβ (at 1×, 2× and 5× of 43 nM) was added, and samples were incubated for another 10 min. The DNA–protein complexes were separated from free DNA substrate using 6% native PAGE. The black dot marks the multiple binding of MutSβ to the Tdp-ICL.
Figure 5.
Figure 5.
Independent binding of MutSβ and XPC–RAD23B to Tdp-ICLs at low (Kapp) protein concentrations. (A) EMSA analysis of purified human recombinant MutSβ (43 nM) and XPC–RAD23B (6.5 nM) with Tdp-ICLs (10 nM). An α-MBP antibody was used to supershift the XPC–RAD23B–Tdp-ICL complex (by identification of the MBP-tagged XPC protein). Lane 1, DNA substrate; lane 2, XPC–RAD23B; lane 3, XPC–RAD23B and α-MBP antibody; lane 4, MutSβ; lane 5, XPC–RAD23B and MutSβ; lane 6, XPC–RAD23B, α-MBP antibody and MutSβ. The star marks the protein–DNA complex shifted by an α-MBP antibody. (B) Southwestern blot analysis of the DNA-protein complexes and unbound proteins using 4% native PAGE. The gels were exposed to X-ray film and then transferred to a PVDF membrane. The membrane was probed with the α-MSH2 antibody and then stripped and blotted again with the α-MBP antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akkari YM, Bateman RL, Reifsteck CA, Olson SB, Grompe M. DNA replication is required to elicit cellular responses to psoralen-induced DNA interstrand cross-links. Mol. Cell. Biol. 2000;20:8283–8289. - PMC - PubMed
    1. Dronkert ML, Kanaar R. Repair of DNA interstrand cross-links. Mutat. Res. 2001;486:217–247. - PubMed
    1. McHugh PJ, Spanswick VJ, Hartley JA. Repair of DNA interstrand crosslinks: molecular mechanisms and clinical relevance. Lancet Oncol. 2001;2:483–490. - PubMed
    1. Momtaz K, Fitzpatrick TB. The benefits and risks of long-term PUVA photochemotherapy. Dermatol. Clin. 1998;16:227–234. - PubMed
    1. Jain A, Wang G, Vasquez KM. DNA triple helices: Biological consequences and therapeutic potential. Biochimie. 2008;90:1117–1130. - PMC - PubMed

Publication types