NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

Abstract

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA-XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.

Figure 1

The ¹H–¹⁵N HSQC spectrum of RPA-AB, 25°C. Most of the amide resonances have been assigned. Two resonances of amino side chains are represented by lines. The amide resonances from the histidine tag are represented by the hash symbol.

Figure 2

Comparison of the ¹H–¹⁵N HSQC spectra of RPA-A in the absence (black) and presence of TT-10 (blue) or CPD-10 (red), 27°C.

Figure 3

The average chemical shift changes (Δδ_avg) in the ¹H and ¹⁵N resonances of RPA-AB upon addition of TT-10 and CPD-10. The residues whose cross-peaks disappear upon the addition of ssDNA are represented as dots.

Figure 4

(A) Comparison of the ¹H–¹⁵N HSQC peaks of S223, E232, Q268 and N274 of RPA-A in the absence (black) and presence of the TT-10 (blue) or CPD-10 (red), 27°C. (B) Comparison of the ¹H–¹⁵N HSQC peaks of I332, V356, V383 and L394 of RPA-AB in the absence (black) and presence of the TT-10 (blue) or CPD-10 (red).

Figure 5

Mapping of the ¹⁵N-labeled RPA-AB residues with the chemical shift perturbation effect observed in the ¹⁵N–¹H HSQC spectra. Residues with the largest chemical shift changes (Δδ_avg > 0.07 p.p.m.) following titration with ssDNA are indicated in blue. Residues with the largest differences in the chemical shift changes (Δδ_avg > 0.04 p.p.m.) observed with TT-10 versus CPD-10 are indicated in red. The four residues that have aromatic side chains involved in stacking interactions with bases in the ssDNA are represented in green.

Figure 6

(A) Binding of RPA-A to the TT/AA-10 duplex as a function of DNA concentration in the absence (upper) and presence (lower) of equimolar XPA-MBD. Populations of RPA-A binding to the TT-10 and AA-10 strands are represented by white and black bars, respectively. (B) Binding of RPA-A to the CPD/AA-10 duplex as a function of DNA concentration in the absence (upper) and presence (lower) of equimolar XPA-MBD. Populations of RPA-A binding to the CPD-10 and AA-10 strands are represented by white and black bars, respectively. (C) Binding of RPA-AB to the CPD/AA-10 duplex as a function of DNA concentration in the absence (upper) and presence (lower) of equimolar XPA-MBD. Populations of RPA-AB binding to the CPD-10 and AA-10 strands are represented by white and black bars, respectively.

Figure 7

Binding of RPA-A to the CPD/AA-10 duplex as a function of the concentration of XPA-MBD. Populations of RPA-A binding to the CPD-10 and AA-10 strands are represented by white and black bars, respectively. The molar ratio of the DNA duplex and RPA-A is 0.3.

Figure 8

Model for the molecular mechanism of damage recognition and verification in GGR. XPC-hHR23B first recognizes the site where the helix distortion is induced. RPA, XPA and TFIIH then may be recruited to the suspected site of damage to verify the presence of a lesion. If there is a lesion (left), RPA binds to the undamaged DNA strand, and XPA binds to the damaged strand specifically and interacts with TFIIH. The pre-incision complex containing the fully opened DNA would then be assembled. If there is no lesion (right), the strand-specific binding of RPA and XPA proteins to DNA does not occur, and the NER process is aborted.