JUST days before the Dutch referendum on the proposed European constitution, a local political sociologist forecast a victory for the Noes. People were likely to reject Euro-initiatives, he argued, when they were angry about immigration, pessimistic about the economy, and hostile to the current government. All three factors were present in Holland. So ...

As it happened, the Noes did more than prevail; they won in a two-to-one landslide. It was a result that shocked the Dutch establishment, which had almost unanimously backed the constitution. The Euro-establishment across the continent, equally thunderstruck, has argued in mitigation that the Dutch voters did not really know what they were doing: They were not rejecting Europe but reacting to local political discontents.

At first glance the sociologist's prediction might seem to support this defense. A closer look, however, suggests that the Dutch were following a deep social instinct: They sensed that "Europe" was somehow connected to their other discontents, and were striking the only blow available to them. What links the EU constitution to anti-government sentiment, immigration, and economic pessimism is voter anger with elite-driven politics. European integration is generally admitted to be an elite project--the current cant phrase that it needs to be better "explained" to the masses makes that embarrassingly clear. High levels of (mainly Muslim) immigration and accompanying policies of multiculturalism are similarly regarded by elites as necessary steps towards a more diverse society.

Most ordinary Dutch people were either opposed to or apathetic towards these social experiments. But the major parties favored them and the media preached them. That left voters with no obvious avenues of protest. And the issues did not intrude on their lives to the extent of rousing them to rebellion--until two atrocities occurred. Pim Fortuyn, the gay libertarian leader of a quirky anti-immigration party, was murdered by an assailant who cited Fortuyn's opposition to large-scale Muslim immigration as his justification. Next, filmmaker Theo van Gogh was murdered by a Muslim extremist who objected to a film he had made attacking Islam for its hostility to women's rights. These two murders sharpened the sense of many Dutch people that their country was being transformed in ways they disliked--and also made them unsafe.

FINGERS IN THE DIKE

Not only conservatives and traditionalists felt these fears. Liberals, feminists, and libertarians like Fortuyn who prized the tolerance, sexual equality, and libertinism of Dutch society were also made nervous. Unpolitical Dutch people voted with their feet. There had long been a "white flight" from schools and urban neighborhoods with large numbers of immigrants; now the Dutch began to emigrate in large numbers abroad.

The political effects of this pessimism were dramatic. The Fortuyn party made major gains in successive elections; the establishment conservative and liberal parties adopted many of its policies; and when the Euro-referendum came along, the Dutch voted no. They were rejecting two things--a constitution that transferred many more powers, including immigration control, from the Dutch parliament to the impenetrable committees of the EU, and a style of bureaucratic politics that was haughtily undemocratic and deaf to their concerns. Those concerns can be very simply summarized: How do we deal with a large and growing Muslim minority that contains Islamists who hate Dutch society and are prepared to use violence against it--and therefore us? Dutch Muslims, for their part, have concerns rooted in the same situation: How do we deal with the fact that we are distrusted and discriminated against by our neighbors because of the actions of a minority of radical hotheads?

The facts underlying these questions are relatively straightforward. Holland's Muslims account for about 15 percent of its population. In cities such as Rotterdam, where immigrants have tended to congregate, the Muslim proportion is about one-third of the local population. And these percentages are rising. Estimates of the number of (generally young male) Muslims who are actively sympathetic to Islamism are inevitably more speculative. The usual guess is that 15 percent of the Muslim population is sympathetic to Islamism and a much smaller percentage, say 4 percent, actively so. In a growing immigrant population, however, these low percentages translate into quite large numbers of potential terrorists.

What makes these possibilities so unsettling to the law-abiding Dutch is that the law is ill-equipped to deal with the threat. In 2003, Dutch intelligence discovered ties between a group of 40 to 50 North African radicals in Holland and a terrorist network that had carried out the Casablanca bombings of that year. (Some were arrested, but because intelligence evidence is not permitted in Dutch courts, they had to be released.) One of the group was Mohammed Bouyeri, who subsequently murdered van Gogh.

To elucidate how human DNA polymerase β (pol β) discriminates dATP from dCTP when processing 8-oxoguanine (8-oxoG), we analyze a series of dynamics simulations before and after the chemical step with dATP and dCTP opposite an 8-oxoG template started from partially open complexes of pol β. Analyses reveal that the thumb closing of pol β before chemistry is hampered when the incorrect nucleotide dATP is bound opposite 8-oxoG; the unfavorable interaction between active-site residue Tyr^sup 271^ and dATP that causes an anti to syn change in the 8-oxoG (syn):dATP complex explains this slow motion, in contrast to the 8-oxoG (anti):dCTP system. Such differences in conformational pathways before chemistry for mismatched versus matched complexes help explain the preference for correct insertion across 8-oxoG by pol β. Together with reference studies with a nonlesioned G template, we propose that 8-oxoG leads to lower efficiency in β's incorporation of dCTP compared with G by affecting the requisite active-site geometry for the chemical reaction before chemistry. Furthermore, because the active site is far from ready for the chemical reaction after partial closing or even full thumb closing, we suggest that pol β is tightly controlled not only by the chemical step but also by a closely related requirement for subtle active-site rearrangements after thumb movement but before chemistry.

DNA base and sugar lesions caused by cellular reactive oxygen species contribute to mutagenesis and cardnogenesis (1). One of the most prevalent lesions in the genome is 7, 8-dihydro-8-oxoguanine (8-oxoG) (2) (Fig. 1 b), in which C8 is oxidized to a carbonyl group and N7 is transformed to an -NH- group. The 8-oxoG nucleoside can adopt two glycosidic conformations: the ami conformation when paired with a complementary C (Fig. 1 c), and the syn conformation when paired with A to form a Hoogsteen basepair (Fig. 1 a); the latter avoids a clash between the deoxyribose backbone and the 8-oxo group. When it is not repaired, the mismatched 8-oxoG:A basepair can introduce G:C to T:A transversion mutations (i.e., the mispairing of 8-oxoG with A will result in thymine in the next replication cycle). Such mutations contribute significantly to somatic mutations associated with spontaneous cell transformations (3-6).

High- and low-fidelity DNA polymerases, such as human DNA polymerase β and Sulfoiobus solfataricus P2 DNA polymerase IV (Dpo4), often encounter 8-oxoG (7-11) and can bypass it in the replication or repair process. Steady-state kinetic data suggest that pol β prefers dCTP over dATP incorporation by twofold and that dCTP insertion is only threefold less efficient opposite 8-oxoG than opposite a normal dG( 12).

A partial explanation to the accommodation of 8-oxoG: dCTP comes from the ternary crystal structure of the pol β/ substrate complex with 8-oxoG (13): the structure shows that the matched 8-oxoG (anti):dCTP basepair is easily tolerated by pol β due to a 184° flip of the template backbone (O3'-P-O5'-C5'), thereby avoiding the clash between O8 and O5' and O1P of 8-oxoG. The mismatched 8-oxoG (syn):dATP basepair, in contrast, is not as stable as the matched pair in pol β. Crystallography has not captured the syn conformation of 8-oxoG pairing with dATP in pol β but rather captured ami 8-oxoG stacking with dAMP (12).

Besides the crystal structures of pol β 8-oxoG complexes, other DNA polymerases across different families with 8-oxoG at the template position have also been resolved crystallographically, These include RB69 from the B-family (10), T7 DNA polymerase (14,15) and Bacillus fragment (BF) from the A-family (11), and Dpo4 from the Y-family (16). However, none of these wild-type enzyme complexes captured syn 8-oxoG pairing with dATP at the active site. Recently, only by mutating Lys^sup 536^ to Ala has the ternary form of T7 DNA polymerase resolved the 8-oxoG (syn):dATP basepair (15).

Kinetic data for pol β, RB69. Dpo4, wild-type T7, LyS^sup 536^ Ala T7, and BF (10-12,15,16) indicate preference ratios of dCTP over dATP incorporation opposite 8-oxoG to be 2:1.20:1, ~70:1.2:1,1:20, and 1:9, respectively. We thus can infer that the incorporation of 8-oxoG (iyn):dATP mismatch is energetically Jess favorable in pol β, wild-type T7. RB69, BF, and Dpo4, but more favorable in LyS^sup 536^ Ala T7, than the corresponding 8-oxoG (anti):dCTP basepair.

Although the nascent basepair 8-oxoG:dAMP is distorted in the pol β/DNA crystal structure, a modeling of dATP pairing with syn 8-oxoG in the closed pol β revealed no serious steric clashes at the active site (17). Since pol β maintains fidelity in DNA replication through an inducedfit mechanism-the thumb subdomain of pol β closes when the correct nucleotide complementary to the template residue binds (18-22) and opens after the chemical reaction to release the reaction products and translocate DNA-the induced-fit cycle may also contribute to differentiating dATP and dCTP insertions opposite 8-oxoG and dG (12). Specifically, dATP is less efficiently inserted opposite 8-oxoG than dCTP in pol β, though dATP insertion is ~10^sup 5^ more efficient compared with it opposite dG. Furthermore, computational studies of pol β (22-28) have shown that conformational pathways of pol β before and after the chemical reaction play a vital role in determining its fidelity. With the ultimate goal of understanding the fidelity mechanism of pol β in processing 8-oxoG, we investigate here the dynamic process of pol β's conformational pathways before and after the chemical reaction by performing a series of six dynamics simulations starting from intermediate (partially open) structures where the incoming nucleolides dCTP and dATP pair with 8-oxoG in anti or syn orientations, Although insertion rates of dATP and dCTP opposite 8-oxoG in pol β differ slightly, atomic-level simulations can help unravel systematic differences in their conformational pathways to explain biological observations. In fact, our transition path sampling simulations further dissected the conformational and energetics pathways of correct and incorrect nucleotide insertions opposite 8-oxoG in pol β (29). The resolved free energy barriers in that work along with the results here suggest that the different transition states and sequences of conformational events during thumb closing for the two systems could be correlated to different stabilities of the respective closed states and associated insertion efficiencies.