The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

Background-Clinical and radiologic predictors of cerebral infarction occurrence and location after aneurysmal subarachnoid hemorrhage have been seldom studied. On multivariable analysis, only presence of symptoms ascribed to vasospasm (PϽ0.01) and evidence of vasospasm on TCD or angiogram predicted cerebral infarction (PϽ0.01). TCD and angiogram agreed on the diagnosis of vasospasm in 73% of cases (95% CI, 63% to 81%), but the diagnostic accuracy of this combination of tests was suboptimal for the prediction of cerebral infarction occurrence (sensitivity, 0.72; specificity, 0.68; positive predictive value, 0.67; negative predictive value, 0.72). Location of the cerebral infarction on delayed CT was predicted by neurological symptoms in 74%, by aneurysm location in 77%, and by angiographic vasospasm in 67%. Conclusions-Evidence of vasospasm on TCD and angiogram is predictive of cerebral infarction on CT scan but sensitivity and specificity are suboptimal. Cerebral infarction location cannot be predicted in one quarter to one third of patients by any of the studied clinical or radiological variables. (Stroke.

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.The problem of enzymatic detection of a single damaged base in the context of a vast genome of nearly isomorphous undamaged bases has intrigued the DNA repair community virtually since the discovery of the DNA base excision repair pathway (1). The initiating step in this pathway begins with the enzymatic hydrolysis of the glycosidic bond that attaches the damaged base to the deoxyribose phosphate DNA backbone, setting the stage for the multistep base excision repair process to begin (Fig. 1). The cellular sentinels at this first step are the remarkable DNA glycosylase enzymes (2). Although these enzymes fall into different structural classes, and each is specialized for the detection and removal of different types of damaged bases (Table 1), with the sole exception of pyrimidine dimer DNA glycosylase (3), these enzymes have converged on a single mechanistic solution for damaged base recognition and excision: rotation of the damaged base from the DNA base stack into a sequestered active site pocket where chemistry occurs (Fig. 2). This process has been called either base or nucleotide "flipping" by various investigators (4,5), and connects the damage encounter event with the catalytic step of bond scission.Base flipping involves one of the most extended and improbable reaction trajectories in biology. The overall reaction is driven forward solely by the use of enzyme binding energy for DNA, which is used to pay for the significant energetic costs of extracting a base from the DNA base stack (11). These costs include breaking of Watson-Crick hydrogen bonds, the disruption of aromatic stacking interactions with adjacent bases, and large perturbations in the phosphate torsion angles around the flipped base. Initial structural investigations into † This work was supported by NIH grant GM056834 (J.T.S).

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