DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion

Abstract

HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G↓CGC in DNA. We report three structures of HinP1I–DNA complexes: in the presence of Ca2+ (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg2+ (post-reactive complex). HinP1I forms a back-to-back dimer with two active sites and two DNA duplexes bound on the outer surfaces of the dimer facing away from each other. The 10 bp DNA duplexes undergo protein-induced distortions exhibiting features of A-, B- and Z-conformations: bending on one side (by intercalation of a phenylalanine side chain into the major groove), base flipping on the other side of the recognition site (by expanding the step rise distance of the local base pair to Z-form) and a local A-form conformation between the two central C:G base pairs of the recognition site (by binding of the N-terminal helix in the minor groove). In the pre- and post-reactive complexes, two metals (Ca2+ or Mg2+) are found in the active site. The enzyme appears to cleave DNA sequentially, hydrolyzing first one DNA strand, as seen in the post-reactive complex in the crystalline state, and then the other, as supported by the observation that, in solution, a nicked DNA intermediate accumulates before linearization.

Mechanism of MutS Searching for DNA Mismatches and Signaling Repair

“…Specifically, to flip out a base at a GT mismatch requires more energy than to flip out an unpaired T because hydrogen bonds between the G and T must be broken, whereas no hydrogen bonds are required to be broken for an unpaired T. Consequently, the additional energy gained from the hydrogen bond between the Glu and DNA may be more important for mismatches than for IDLs. The observation that Phe 39 is absolutely essential for formation of the unbent URC is also consistent with the suggestion of base flipping because Phe is commonly used by proteins that flip out bases (32,33,35,45,46). For instance, for N-6 adenine DNA methyltransferase M-TaqI, Phe…”

“…, which makes an edge-to-face interaction with the extruded adenine, has been shown to significantly stabilize the base in the extrahelical position (32,46). Flipping of the mismatched base in the URC by MutS is consistent with both in vitro and in vivo studies of DNA MMR (3).…”

“…In addition, the loss of specificity, taken together with the wild type-like DNA bending, suggests that the relative ease of bending of mismatches compared with homoduplex DNA does not contribute a significant amount of stability to the MutS mismatch complex. The observation that Phe 39 is not necessary for MutSinduced DNA bending is quite surprising, given that phenylalanines are generally involved in inducing DNA bending (32)(33)(34)(35). Notably, it is the unbent population that is absent from the specific complexes formed with MutS-F39A (Fig.…”

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

“…Also, a few examples in the recent literature have elucidated localized dynamics in DNA interac-tion enzymes (31)(32)(33), including cases of rigidification (33) and flexibility (34) of the nucleic acid on binding. High-temperature factors, or the absence of interpretable data, are often diagnosed as disorder.…”

SssI DNS metiltranszferáz által katalizált citozin dezamináció

“…A felismerési komplexum aszimmetriája azt sugallja, hogy ezek az enzimek nickelő enzimként működnek, és a kettősszálú DNS hasítást tulajdonképpen két egymás utáni egyszálú hasítás (nick) révén érik el. Ez a működési mód eddig a HinPI és az MvaI endonukleázoknál bizonyított, sőt ez utóbbi esetén azt is kimutatták, hogy képes különbséget tenni a két szál közt, az A-közepű szálat gyorsabban hasítva (Horton, 2006;Kubareva és mtsai., 1991 Ennek a helyes kialakítása és fenntartása elengedhetetlen a szervezet normális működéséhez (Jeltsch, 2002;Jones, 2001 The genomic distribution of the methyl cytosine residues forms a pattern, which varies among species, and it is tissue specific. Properly established and maintained methylation patterns are essential for mammalian development and for normal function of the organism.…”