The mechanisms by which NEIL and NEIL excise bulky adducts
The mechanisms by which NEIL1 and NEIL3 excise bulky adducts and unhook ICLs are unclear. Structures of NEIL1 are available only in complex with DNA containing small lesions 79., 80., 81., and the single available structure of NEIL3 lacks DNA (Figure 5D,E) . Nonetheless, comparison of the two structures, as well as structures of the bacterial enzymes, provides a basis for speculation. Both NEIL1 and NEIL3 share the same structural architecture and arrangement of N-terminal catalytic residues (Pro2/Val2 and Glu3) as Fpg and Nei. Like the bacterial enzymes , NEIL1 also utilizes three intercalating residues to stabilize the extrahelical conformation of the DNA substrate (Figures 2B and 5D). The capping loop in NEIL1, which contacts the flipped lesion in the bacterial enzymes, is similar to that of Nei 9., 79., 80., 81.. This loop is largely disordered in all structures of Nei and most structures of NEIL1 (Figure 5D), suggesting an inherent flexibility that may permit recognition and excision of diverse lesions. In Necrostatin 1 to the well-ordered loop in Fpg , which contacts extrahelical nucleobases through amide groups in the protein backbone (Figure 2B,C), the partially disordered loop in NEIL1 interacts with the lesion through the sidechain of a conserved arginine residue . The flexibility of this loop may enable expansion of the nucleobase binding pocket to accommodate bulky adducts, such as nitrogen mustard and aflatoxin B1 derivatives of N6-(2′-deoxyribosyl)-2,6-diamino-4-oxo-5-formamidopyrimidine (FapyG) and psoralen monoadducts (Figure 5A) 56., 74.. This flexibility may also allow NEIL1 to bind to triplex substrates by pulling the short, third strand away from the duplex and binding the duplex and the crosslink in the same manner as a bulky adduct in double-stranded DNA.
By contrast, NEIL3 shares fewer structural features with the bacterial enzymes 9., 73.. Unlike Fpg and Nei, NEIL3 appears to lack intercalating residues, with the possible exception of Met99 (Figure 5E) . In NEIL1 and the bacterial enzymes, the two other intercalating residues primarily interact with the complementary DNA strand in duplex substrates 9., 81.. The absence of these residues is consistent with the established preference of NEIL3 for lesions in single-stranded DNA 71., 73., and suggests that NEIL3, like AlkZ, may unhook ICLs using a non-base-flipping mechanism. However, the non-base-flipping mechanisms employed by AlkZ and AlkD/YtkR2 limit these proteins to excising positively charged lesions, which do not require activation of the nucleobase through contacts in the nucleobase binding pocket (Box 1). NEIL3 acts on neutral lesions and crosslinks 54., 56., 62., which would seemingly require activation by the enzyme. Nevertheless, the capping loop that performs this function in NEIL1, Nei, and Fpg is strikingly short in NEIL3, and seems unlikely to contact even a fully flipped nucleotide (Figure 5E). Although such an open nucleobase binding pocket is consistent with removal of bulky adducts, including nitrogen mustard derivatives of FapyG , how NEIL3 activates neutral lesions for excision is unclear, and will require additional structural experiments with appropriate DNA substrates. Similarly, mechanistic understanding of NEIL2 has been severely limited by a lack of available structural information. However, the amino acid sequence of NEIL2 shares all features of NEIL3 that seem to enable unhooking of ICLs, and NEIL2, like NEIL3, also shares a preference for nonduplex structures . Based on these common features and the interaction of NEIL2 with RNA polymerase II , we speculate NEIL2 may be involved in transcription-coupled repair of ICLs.
Protection from Foreign DNA Restriction-modification systems protect prokaryotes from the potentially deleterious effects of foreign DNA, regulating genetic exchange among bacteria and guarding against infection by bacteriophages 83., 84., 85.. At a minimum, these systems comprise two components: a restriction endonuclease that recognizes a specific DNA sequence and introduces one or more strand breaks, and a DNA methyltransferase that modifies the same sequence to render it resistant to the endonuclease. Type II restriction endonucleases use a Mg2+-dependent mechanism to cleave both strands of DNA, usually within a short palindromic sequence, producing a DSB . However, genomic analyses recently identified a novel type II enzyme in hyperthermophilic archaea – with homologs in both thermophilic and mesophilic bacteria – that generates strand breaks without requiring Mg2+ 87., 88., 89.. Unlike other type II enzymes, which directly hydrolyze the phosphodiester backbone, R.PabI hydrolyzes the glycosidic bond of 2′-deoxyadenosine, producing nearby AP sites on opposing strands. A DSB is then generated by heat-promoted β-elimination or enzymatic AP endonuclease or lyase activities (Figure 6A) 90., 91., 92.. Similarly to restriction endonucleases, R.PabI cuts exclusively within palindromic GTAC sequences, making it the only known sequence-specific DNA glycosylase. To avoid formation of DSBs in the genome, R.PabI is coexpressed with the DNA methyltransferase M.PabI, which forms N6-methyl-2′-deoxyadenosine (N6-mA) within the same GTAC sequences, preventing excision by R.PabI 91., 93..