DNA-PROTEIN CROSSLINKS

Kendric C. Smith1 and Martin D. Shetlar2

1Stanford University School of Medicine
927 Mears Court, Stanford, CA 94305-1041
kendric@stanford.edu
www.stanford.edu/~kendric/

2Department of Pharmaceutical Chemistry
Box 0446, 513 Parnassus Ave, Med Sci 920
University of California San Francisco
San Francisco, CA 94143-0446
shetlar@cgl.ucsf.edu

Introduction

Nucleic acids in living cells are associated with a large variety of proteins. Therefore, it is logical to assume that the ultraviolet (UV) irradiation of cells could lead to reactive interactions between DNA and the proteins that are in contact with it. One such reaction that can be envisioned is that the amino acids in these associated proteins may become crosslinked to the bases in DNA. Indeed such reactions do occur, and appear to be important processes that photoexcited DNA undergoes in vivo, as well as in DNA-protein complexes in vitro.

The first example of UV-induced crosslinking of DNA and proteins in a living system (Escherichia coli) was reported in 1962 (Smith, 1962). In the same study, it was noted that the treatment of E. coli with acridine orange and visible light also resulted in DNA-protein crosslinking. In fact, on the basis of the dose of radiation needed to produce the same reduction in colony formation, it was found that visible light plus dye crosslinked a larger percentage of the DNA than did UV radiation. On the other hand, X-irradiation produced little if any crosslinking (Smith, 1962). Since these early studies, photoinduced DNA-protein crosslinking has been observed in other cellular systems, as well as in isolated DNA-protein complexes. Among the latter are the crosslinking of histones to DNA in eukaryotic nucleosomes, the crosslinking of RNA polymerase and DNA polymerases to DNA, and the cross-linking of the gene 5 "melting" protein from fd phage to single-stranded DNA. (For reviews and references, see Smith,1976; Shetlar,1980; Saito and Sugiyama,1990).

Detecting DNA-Protein Crosslinks

A number of methods have been developed for detecting DNA-protein cross-links (reviewed in Shetlar, 1980). The first method used was based upon the extraction of DNA free of protein from cells following UV irradiation. DNA can be isolated from E. coli free of protein using a detergent (sodium dodecyl sulfate) extraction procedure (Smith and Kaplan, 1961; Smith, 1962), however, with increasing doses of UV radiation there is a linear decrease in the amount of free DNA that can be extracted, and an increasing amount of DNA that remains associated with the denatured proteins (Figure 1).

Figure 1. The extractability of DNA from E. coli B/r free of protein following increasing doses of UV radiation (254 nm). For comparison, the rate of loss of thymine due to the formation of thymine dimers is plotted.
[Modified from Smith, 1962]

Note that 30% of the DNA is seven times more sensitive to crosslinking with protein than is the remainder (see below). At a UV dose that kills 99% of the cells, about 10% of the DNA was crosslinked with protein (Smith, 1962).

As one might predict, it is the replicating portion of the DNA chromosome that is more sensitive to UV-induced crosslinking with protein (Smith, 1964a). This was tested by pulse labeling a log-phase culture of E. coli with tritiated thymine, and then replacing the radioactive thymine with non-radioactive thymine, and allowing the culture to continue growing exponentially. Samples were taken at various times. One sample was UV-irradiated (254 nm) and one was not, and then DNA was extracted from both. DNA-protein crosslinking was greatest immediately after the labeling, and one generation time after the labeling, i.e., at the time when the labeled section of DNA was again being replicated. The experiment was followed for 2 generation times.

Photoreactions of Nucleobases and Nucleosides with Amino Acids and Related Compounds

The first amino acid shown to photochemically add to uracil was cysteine, to form 5-S-cysteinyl-6-hydrouracil (Smith and Aplin, 1966). The structure of the mixed photoproduct of thymine and cysteine was also determined (Smith, 1970) (Figure 2). Later work showed that this compound is photochemically-produced in two diastereomeric forms, and that two other compounds, namely 5-S-cysteinylmethyluracil (Varghese, 1973) and 5-S-cysteinyl-5,6-dihydrothymine (Shetlar and Hom, 1987) are also produced when thymine is irradiated in the presence of cysteine.

Figure 2. Adducts arising from the photoreactions of thymine with cysteine (left) and N-acetyltyrosine (right).
R = CH₂CH(NH₂)COOH

The photoreactivity of various polynucleotides for the addition of [35S]cysteine was also studied. Rate constants for this reaction were measured; poly rU was found to be the most reactive (k =21.8), followed by poly rC (k =8.1), poly dT (k =5.4), and poly rA (k =0.6). Ribonucleic acid showed a biphasic response with k =21.8 and k =4.8 (Smith and Meun, 1968).

In addition to the products of the photoreactions of thymine and uracil with cysteine, photoproducts have been characterized in a number of other systems containing nucleobases (or nucleosides), and amino acids (or amino acid analogs) (reviewed in Saito and Sugiyama, 1990). For example, the reaction of thymine with the phenolic ring system contained in N-acetyltyrosine results in a compound with the structure shown in Figure 2 (Shaw et al., 1998), while the reaction of thymidine with lysine yields a photoproduct of a very different nature (Figure 3) (Saito et al., 1981, 1983a). The production of this compound involves the attack of the ε-amino moiety of the lysine on the carbonyl group in the 2-position of thymidine. There is evidence that cytosine and 5-methyl-cytosine react with lysine to form adducts of a similar nature (Dorwin et al., 1988).

Figure 3. Structure of the photoproduct arising from the reaction of thymidine with lysine. R1 is (CH2)4CH(NH2)COOH, and R2 is a 2'-deoxyribosyl moiety.

Other amino acids are also reactive with nucleobases and polynucleotides. The first survey performed determined the ability of the 22 common amino acids to add photochemically (254 nm) to uracil. The 11 reactive amino acids were glycine, serine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, histidine, arginine and lysine. The most reactive amino acids were phenylalanine, tyrosine and cysteine. Therefore, the photochemical addition of amino acids to uracil appears to be a fairly common phenomenon (Smith, 1969). A later study of the photoreactivity of polyuridylic acid indicated that all 20 amino acids were reactive, as well as a variety of glycylpeptides and other peptides (Shetlar et al., 1984c).

When thymine was similarly screened for photochemical reactivity with 22 common amino acids, only lysine, arginine, cysteine and cystine formed heteroadducts after exposure times similar to those used for uracil. Thymine was generally less reactive than uracil with amino acids when exposed to UV radiation (Schott and Shetlar, 1974). This may be due to the by shielding of carbon-5 in thymine by the methyl group at that position (Figure 2).

Another study used a fluorescence assay method to assess the photoreactivity of DNA and polynucleotides for the addition of various amino acids and peptides. The reactivities of the 20 amino acids commonly occurring in proteins were determined for their photochemical addition to denatured calf thymus DNA at pH 7. Fifteen amino acids were reactive, with cysteine, lysine, phenylalanine, tryptophan, and tyrosine being the most reactive. Alanine, aspartic acid, glutamic acid, serine, and threonine were unreactive (Shetlar et al., 1984a). Corresponding quantum yields were also determined for many of the glycyl dipeptides (e.g., glycyl serine) of the same amino acids. It was found that of the peptides studied, those containing lysine, cystine, proline, histidine and the various aromatic acids (phenylalanine, tyrosine, tryptophan) were the most reactive (glycyl cysteine was not studied). Interestingly, in peptide form, all of the amino acids studied displayed some degree of reactivity. In almost all cases, amino acids incorporated into peptides had higher reactivities towards photoaddition than the corresponding carboxyl terminal amino acids at the same concentration.

Measurements similar to those done on DNA photoreactivity were made on the photochemical reactivity of four polyribonucleotides, namely poly rA, poly rC, poly rG acid, and poly rT, towards the addition of glycine and the L-amino acids commonly occurring in proteins, excluding proline (Shetlar et al., 1984b). Poly rA was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine being the most reactive. Poly rG reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Poly rC showed photoreactivity with fifteen amino acids, with phenylalanine, lysine, cysteine, tyrosine and arginine having the highest reactivities. Poly rT was reactive with fifteen of nineteen amino acids surveyed, and showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid. Studies on the photoaddition of various glycyl dipeptides with each of the polynucleotides indicated that they were often more reactive than the amino acids themselves. In general, poly rT was the most reactive polynucleotide towards photoaddition for most of the amino acids and peptides studied. For example, the quantum yields for the photoaddition of phenylalanine to poly rT, poly rC, poly rA, and poly rG were in the ratio of 80:4:5:3.

Bromo- and iodo-substituted uracils and cytosines are also capable of photoreaction with amino acids. For example, 5-bromouracil photocouples with tyrosine, tryptophan and histidine (Dietz and Koch, 1987), as well as peptide linkages (Dietz et al., 1987). 5-Bromouracil also reacts with ethylamine (Shetlar et al., 1991), a lysine analog, to form a compound analogous to that formed in the reaction of thymine with lysine (Figure 3).

Cells of E. coli whose thymine has been replaced by 5-bromouracil are more sensitive to killing by UV irradiation than are unsubsititued cells (Greer, 1960; Kaplan et al., 1962), and they show a 5-fold greater sensitivity of UV-induced DNA-protein crosslinking than do unsubstituited cells (Smith, 1964b).


Photo-induced Crosslinks in DNA-Protein and Related Systems

A number of nucleobase-amino acid crosslinks have been identified in various UV-irradiated DNA-protein and related systems. For example, thymine-lysine conjugates have been identified as participants in the crosslinking in DNA-histone systems (Saito et al., 1983b; Kurochkina et al., 1987). Thymine-cysteine conjugates have been shown to be produced in the UV-induced crosslinking of the gene 5 protein of fd phage to its corresponding DNA (Paradiso and Konigsberg, 1982). At the level of a nucleoside-peptide system, the single tyrosine contained in Angiotensin I was crosslinked to thymidine when a solution containing these two compoents was irradiated (Shaw et al., 1992). Similar results were obtained when thymidine, thymidine-5'-phosphate and thymidylyl-[3'-5']-2'-deoxyadenosine were irradiated in the presence of the tyrosine-containing heptad repeat peptide unit found in the largest subunit of the eukaryotic RNA polymerase II multiprotein complex (Connor et al., 1998a).


Photocrosslinking as a Tool for Structural Studies of DNA-Protein Complexes

DNA-protein crosslinking is a valuable tool for studying the structure of DNA-protein complexes. Since the only amino acids and nucleobases that can participate in crosslinking are those in contact in DNA-protein complexes, crosslinking can potentially be used to identify amino acids (or peptides) and nucleobases in those regions involved in binding protein to DNA. While steady state UV irradiation has been used in many studies, the use of pulsed lasers, in conjunction with mass spectrometry, has provided a powerful alternative approach to studying crosslinking, especially for examining contacts in native DNA-protein complexes. For example, this combination of techniques has been used to identify six crosslinked peptides in the complex formed between the single-stranded DNA binding domain of rat DNA polymerase ß, and the oligonucleotide d(ATATATA) (Connor et al., 1998b). [Experimental aspects of the use of crosslinking to study the structure of nucleic acid-protein complexes are discussed by Williams and Konigsberg (1992) (by steady state UV-induced crosslinking), and by Hockensmith et al. (1992) (by laser pulse-induced crosslinking). Reviews by Meisenheimer and Koch (1997), and Steen and Jensen (2002), provide information about, and references to, a number of studies in which the UV-induced crosslinking of nucleic acid-protein complexes has been used to gain structural information about such complexes.]

DNA-protein complexes in which the DNA component has been modified to contain 5-halogenated uracils or cytosines have also been studied using laser crosslinking-mass spectrometric approaches. For example, it has been shown that tryptophans 54 and 88 in the sequence of the E. coli single-stranded DNA binding protein can be bound to a DNA oligomer in which thymines are replaced with 5-iodouracil moieties (Steen et al., 2001). Nucleic acid-protein complexes, in which thymine has been replaced with 5-bromouracil or cytosine with 5-iodocytosine, have also found use in photochemical experiments designed to study contact regions in these complexes. [For reviews, see Meisenheimer and Koch, 1997; Steen and Jensen, 2002]


Biological Importance of DNA-Protein Crosslinks

Since the crosslinking of DNA and protein by UV radiation is many times more sensitive analytically than is thymine dimer formation, it was suggested that DNA-protein crosslinks may play a significant role in the inactivation of bacteria by UV radiation (Smith, 1962).

This hypothesis was subsequently proven by growing E. coli mutants under different conditions that affect cell sensitivity to UV radiation. A direct correlation was observed between the amount of DNA crosslinked to protein by a given dose of UV radiation, and the intrinsic sensitivity to killing by UV radiation under the several growth conditions studied (Smith et al., 1966).

In addition, the increased sensitivity of E. coli to killing by UV irradiation when frozen, and the variation in this sensitivity as a function of the temperature during irradiation, correlated with changes in the amount of DNA that was crosslinked to protein by UV irradiation. These variations in sensitivity to killing did not correlate with the production of thymine dimers (Smith and O'Leary, 1967).

DNA-protein crosslinks are repaired by postreplication repair (see module on Recombinational DNA Repair), and they cause a longer delay in DNA synthesis than do pyrimidine dimers (Smith and Hamlin, 1977).

These results on the biological importance of UV radiation-induced DNA-protein crosslinks are consistent with the fact that only about 60% of the survival of E. coli after UV irradiation can be photoreactivated (see module on Phototreactivation), i.e., 40% of lethality must be due to lesions other than cyclobutane pyrimidine dimers. DNA-protein crosslinks cannot be photoreactivated (Smith, 1964b).


Summary and Conclusions

DNA-protein crosslinks are important lethal lesions in cells exposed to UV radiation. Crosslinks are particularly disruptive, as they occur mostly in the area of the chromosome that is undergoing replication. The structures of a number of adducts that are potentially responsible for crosslinks formed in UV-irradiated DNA-protein complexes have been determined. Surveys of the reactivity of thymine and uracil, as well as polynucleotides of the DNA and RNA nucleobases towards the photoaddition of amino acids have been conducted. Photocrosslinking is a useful tool to map DNA-protein contacts in DNA-protein complexes.


References

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Connor, DA, Falick, AM, Young, MC, Shetlar, MD. 1998b. Probing the binding region of the single-stranded DNA-binding domain of rat DNA polymerase _ using nanosecond-pulse laser-induced cross-linking and mass spectrometry. Photochem. Photobiol. 68: 299-308.

Dietz, TM, Koch, TH. 1987. Photochemical coupling of 5-bromouracil to tryptophan, tyrosine and histidine, peptide like derivatives in aqueous fluid solution. Photochem. Photobiol. 46: 971-978

Dietz, TM, von Trebra, RJ, Swanson, BJ, Koch, TH. 1987. Photochemical coupling of 5-bromouracil (BU) to a peptide linkage. A model for BU-DNA photocrosslinking. J. Amer. Chem. Soc. 109: 1793-1797

Dorwin, EL, Shaw AA, Hom K, Bethel P, Shetlar, MD. 1988. Photoexchange products of cytosine and 5-methylcytosine with
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Kurochkina, LP, A. A. Komissarov, AA, Kolomiitseva, GY. 1987. Localization of the lysine residue in histone H3 forming a thymine-lysine cross-link when deoxyribonucleo-protein is irradiated with UV light. Biochem. USSR, 52: 1457-1461.

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Smith, KC. 1970. A mixed photoproduct of thymine and cysteine: 5-S-cysteine, 6-hydrothymine. Biochem. Biophys. Res. Commun., 39:1011-1016.

Smith, KC. 1976. The radiation-induced addition of proteins and other molecules to nucleic acids, in Photochemistry and Photobiology of Nucleic Acids, (S.Y. Wang, ed), Academic Press, New York, Vol. II, pp. 187-218.

Smith, KC, Aplin, RT. 1966. A mixed photoproduct of uracil and cysteine (5-S-cysteine-6-hydrouracil). A possible model for the in vivo crosslinking of deoxyribonucleic acid and protein by ultraviolet light. Biochemistry 5:2125-2130.

Smith, KC, Hamlin, C. 1977. DNA synthesis kinetics, cell division delay, and post-repliction repair after UV irradiation of frozen cells of E. coli B/r. Photochem. Photobiol. 25:27-29.

Smith, KC, Hodgkins, B., O'Leary, ME. 1966. The biological importance of ultraviolet light induced DNA-protein crosslinks in Escherichia coli 15 TAU. Biochim. Biophys. Acta 114:1-15.

Smith, KC, Kaplan, HS. 1961. A chromatographic comparison of the nucleic acids from isologous newborn, adult, and neoplastic thymus. Cancer Res. 21:1148-1153.

Smith, KC, Meun, D.H.C. 1968. Kinetics of the photochemical addition of [35S]cysteine to polynucleotides and nucleic acids. Biochemistry 7:1033-1037.

Smith, KC, O'Leary, ME. 1967. Photoinduced DNA-protein cross-links and bacterial killing: A correlation at low-temperatures. Science 155: 1024-1026.

Steen, H, Jensen, ON. 2002, Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry. Mass Spectrometry Reviews 21: 163-182

Steen, H., Peterson, J., Mann, M. , Jensen, ON. 2001. Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA. Protein Sci.10: 1989-2001

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[NOTE: Kendric Smith's papers cited here are available as PDF files.]
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