10 A niversary of IIMCB
10 A niversary of IIMCB
10 A niversary of IIMCB
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
• Filipek R, Potempa J, Bochtler M. A comparison <strong>of</strong><br />
staphostatin B with standard mechanism serine protease<br />
inhibitors. J Biol Chem, 2005; 280:14669-74<br />
Method development<br />
• Chojnowski G, Bochtler M. The statistics <strong>of</strong> the highest E<br />
value. Acta Crystallogr A, 2007; 63:297-305<br />
• Bochtler M, Chojnowski G. The highest reflection<br />
intensity in a resolution shell. Acta Crystallogr A, 2007;<br />
63:146-155<br />
Other<br />
• Breer K, Wielgus-Kutrowska B, Hashimoto M, Hikishima S,<br />
Yokomatsu T, Szczepanowski RH, Bochtler M, Girstun<br />
A, Starón K, Bzowska A. Thermodynamic studies <strong>of</strong><br />
interactions <strong>of</strong> calf spleen PNP with acyclic phosphonate<br />
inhibitors. Nucleic Acids Symp Ser (Oxf), 2008; 52:663-664.<br />
Current Research<br />
Protein-DNA interactions<br />
Two-fold symmetry is a recurrent theme in protein-<br />
DNA interactions. Classic examples are the interactions<br />
between type II restriction endonucleases and their two-fold<br />
symmetric (palindromic) target sequences. In most cases,<br />
two-fold symmetry governs sequence recognition and DNA<br />
cleavage. Exact two-fold symmetry is only possible in DNA<br />
duplexes that consist <strong>of</strong> an even number <strong>of</strong> base pairs. In<br />
duplexes that consist <strong>of</strong> an odd number <strong>of</strong> base pairs, the<br />
requirements <strong>of</strong> hydrogen bonding and two-fold symmetry<br />
conflict for the central base pair. Therefore, such sequences<br />
can at best be pseudosymmetric (pseudopalindromic). At the<br />
center, the possibilities for base recognition are limited: either<br />
the bases in this position are not read out at all, or A:T pairs<br />
(W) are distinguished from G:C pairs (S), irrespective <strong>of</strong> which<br />
DNA strand contains the purine and which strand contains<br />
the pyrimidine base. The latter type <strong>of</strong> recognition poses<br />
difficulties for the “typical” major groove readout <strong>of</strong> the DNA<br />
base sequence: in the major groove, the hydrogen bonding<br />
patterns <strong>of</strong> a G:C and T:A are similar, but differ from the<br />
patterns for C:G and A:T. The research group has systematically<br />
studied restriction endonucleases that recognize and cleave<br />
pseudopalindromic DNA sequences.<br />
The nucleotide flippers Ecl18kI and PspGI: The related<br />
PD-(D/E)XK restriction endonucleases Ecl18kI and PspGI are<br />
specific for the sequences /CCNGG and /CCWGG respectively.<br />
Our crystal structures <strong>of</strong> these enzymes show that both<br />
enzymes form functional dimers that extrude the central<br />
bases <strong>of</strong> their recognition sequences from the DNA and flip<br />
them into pockets <strong>of</strong> the enzymes.<br />
“Nucleotide flips” have been observed before, especially<br />
in the contexts <strong>of</strong> DNA base modification and DNA repair.<br />
However, the nucleotide flips in the complexes <strong>of</strong> Ecl18kI and<br />
PspGI with DNA are unusual in several ways: (a) the enzymes<br />
flip intact bases, which are not modified chemically. (b) the<br />
enzymes flip both bases <strong>of</strong> the DNA stack. (c) the void left<br />
behind by the flipped bases is not filled by DNA intercalating<br />
residues as in most other cases <strong>of</strong> nucleotide flipping, but is<br />
instead closed by “compressing” the DNA, so that the base<br />
pairs that flank the flipped base pair get into almost direct<br />
contact. Ecl18kI simply “skips” the flipped bases for recognition,<br />
but PspGI distinguishes A:T pairs from G:C pairs. Is there a<br />
mechanism that can explain this distinction without the need<br />
to identify individual bases? We reasoned that nucleotide<br />
flipping might serve as a “test” <strong>of</strong> the strength <strong>of</strong> the hydrogen<br />
bonding interactions. According to this model, PspGI would<br />
be “strong” enough to break the two hydrogen bonds that<br />
hold A:T/T:A (“W, weak”), but not G:C/C:G (“S, strong”) pairs<br />
together. Kinetic experiments show that base pair strength<br />
plays a role for base pair discrimination, but is alone insufficient<br />
to explain the high specificity, as was previously concluded for<br />
DNA repair enzymes that recognize lesions in DNA.<br />
The monomeric restriction endonucleases MvaI and<br />
BcnI: The PD-(D/E)XK restriction endonucleases MvaI (CC/<br />
WGG) and BcnI (CC/SGG) recognize similar sequences as<br />
Fig. 1. Co-crystal structures <strong>of</strong> (A) PspGI and (B) Ecl18kI with DNA. The flipped nucleotides are shown in color in all-atom representation. The figure was<br />
taken from Szczepanowski et al., Nucleic Acids Res. 2008, 36:6<strong>10</strong>9-17.<br />
Laboratory <strong>of</strong> Structural Biology 35