Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219)

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with 15N-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA-DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The 15N/1H HSQC spectrum of XPA-EM closely maps onto the 15N/1H HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of 15N/1H HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the 15N/1H HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, 15N-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.     

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group a protein on interaction with DNA intermediates of nucleotide excision repair

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the inter-action of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E ? K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.     

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

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.     

Structural basis for VO<Superscript>2+</Superscript> inhibition of nitrogenase activity (A): <Superscript>31</Superscript>P and <Superscript>23</Superscript>Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

We previously reported the vanadyl hyperfine couplings of VO(2+)-ATP and VO(2+)-ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253-13263, 2002). It was demonstrated that different VO(2+)-nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron-nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal-nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.