Probing the Binding Region of the Single‐Stranded DNA‐Binding Domain of Rat DNA Polymerase β Using Nanosecond‐Pulse Laser‐Induced Cross‐Linking and Mass Spectrometry

Abstract

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.

Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins

“…ESI-MS/MS revealed a nonapeptide bound through tyr133 to a dinucleotide (Golden et al, 1999). Proteolytic products can also be puri®ed by HPLC and analyzed by either ESI-MS (Wong, Pavlovich & Reich, 1998) or MALDI-MS (Connor et al, 1998). These examples were aimed at characterization of photocrosslinked DNA±EcoR1 DNA methyltransferase complexes and probing the ss DNA-binding region of rat DNA polymerase b, respectively.…”

“…More commonly, noncovalent complexes are stabilized by UV (Connor et al, 1998) or chemical (Bastia, 1996) crosslinking prior to analysis, allowing more precise determination of DNA±protein contact points. Recently, basic ®broblast growth factor photocrosslinked with a high af®nity ss 61-mer was treated with trypsin.…”

Characterization of transcription factors by mass spectrometry and the role of SELDI‐MS

“…A different MS approach was used to study the binding of rat DNA polymerase b to single-strand DNA by nanosecond-pulse laser-induced cross-linking and MALDI-MS (Connor et al, 1998). Following cross-linking of the transcription factor-DNA complex, trypsin proteolysis followed by MS analysis revealed six amino acids of polymerase b that were involved in the protein-oligonucleotide conjugate.…”

New interactions between tumor suppressor Fhit protein and a nonhydrolyzable analog of its AP4A substrate

“…Importantly, because the excitation is performed at a relatively long wavelength, other potentially photoreactive chromophores present in a protein are only marginally excited, thus, protein self‐crosslinking can be minimized. To establish the molecular mass of a crosslinked oligonucleotide/protein complex (or oligonucleotides/peptides resulting from enzymatic digests of the crosslinked protein–nucleic acid complex), HPLC coupled with electrospray ionization mass spectrometry (HPLC‐ESI MS = LC‐MS) is a primary method that can be used . Although ESI MS of peptides is performed in the positive ion mode and ESI MS of nucleic acids is normally performed in the negative ion mode, to investigate oligonucleotide/protein complexes, mass spectrometry (MS) can be used in the positive mode.…”