Structure of Escherichia coli AlkA in Complex with Undamaged DNA

Abstract

Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures with that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions.The integrity of covalent structure in the genome is essential for normal cellular function and for faithful transmission of the heritable information reposited therein. DNA inside cells is under constant attack by exogenous environmental toxins and endogenous reactive cellular constituents, giving rise to nucleobase modifications such as oxidation, hydrolytic deamination, and alkylation (1, 2). If left uncorrected, these lesions and the products of their mismanagement by the cell can cause mutations and also interfere with essential cellular processes including transcription, recombination, and DNA replication, events that are causally linked with cancer (3, 4).Although several repair pathways exist for eliminating aberrant nucleobases from the genome, BER 4 is the primary cellular response for the repair of single lesion bases in DNA. The proteins responsible for initiating BER are DNA glycosylases, enzymes that recognize aberrant nucleoside lesions and catalyze scission of their glycosidic linkage. Most BER glycosylases are characterized by high specificity for one particular lesion, or at most a few closely related ones, with this specificity arising from binding interactions between the lesion nucleobase and residues within the enzyme active site (5, 6). There does exist, however, a class of BER enzymes, the 3-methyladenine DNA glycosylases, many members of which exhibit the ability to repair a highly diverse array of nucleoside lesions (7) mostly resulting from DNA alkylation. Prototypical members of this class of enzymes include the Saccharomyces cerevisiae Mag1, human alkyladenine glycosylase AAG, and Escherichia coli 3-methyladenine glycosylase II AlkA. In E. coli, the expression of AlkA is under the control of the adaptive response and the ada regulon, which induces transcription of the alkA gene some hundredfold upon exposure to certain DNA alkylating agents (8 -10). Among the remarkably broad range of AlkA substrates are the alkylated purines N3-and N7-methylguanine and -ad...

Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase

“…The two structures reported in this work show that MutY uses the signature HhH DNA-binding motif common to all HhH superfamily proteins to initiate DNA interactions, indicating that this could be a mechanism employed by all proteins of this superfamily. Such nonspecific DNA contacts by the HhH motif has also been observed in the AlkA undamaged DNA structure (23,33), further suggesting the existence of a common lesion-scanning mechanism for the HhH DNA glycosylases. It is therefore reasonable to assume that the structurally related HhH proteins share a similar mode of interaction all along the base extrusion pathway.…”

“…The fact that the HhH motif accounts for most of the phosphate contacts in the N-LSC suggests that MutY uses it to first engage the DNA in a nonspecific fashion without causing DNA bending or base extrusion. Crystal structure of other HhH-GPD superfamily DNA glycosylases such as AlkA (23,24) and hOGG1, 3 in complex with undamaged DNA also showed a similar mode of interaction, where the HhH motif of the glycosylase interacts with the two phosphate groups that are 3Ј to the target base on the lesion-containing strand.…”

“…Because of the lack of a crystal structure of full-length MutY bound to undamaged DNA, we turned to size exclusion chromatography (SEC)-SAXS to generate a conformational model of the LSC. Previous structures of hOGG1 in complex with undamaged DNA (23,24) 3 have shown that cross-linking the protein to the DNA at particular positions facilitates the extrusion of an undamaged base. To ensure that the target A in the T:A base pair remains fully intrahelical during the course of sample preparation and data collection, we turned to in vitro cleavage assays on disulfide cross-linked MutY complexes having either an oxoG or a T that base pairs with the A at the target site (Fig.…”

“…Crystallization was therefore facilitated by introducing an intermolecular disulfide cross-linker at a site that is 4 base pairs away from the target base pair (12,13,22). The relatively low affinity MutY exhibits toward undamaged DNA (20,23), and the anticipated fleeting nature of such binding led us to introduce a similar cross-linking strategy to obtain a stable and homogenous protein-DNA complex suitable for crystallization. We first set out to crystallize the full-length MutY with undamaged DNA using the same cross-linking site that was used in the LRC (13, 23).…”

Enforced Presentation of an Extrahelical Guanine to the Lesion Recognition Pocket of Human 8-Oxoguanine Glycosylase, hOGG1

“…We have overcome this problem by developing a straightforward means by which to drastically limit the region a protein can explore while diffusing along DNA, through the introduction of an intermolecular disulfide cross-link (DXL) between protein and DNA. This modification has enabled the crystallization and structure determination of multiple protein-DNA complexes (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44).…”