Waters T R, Swann P F
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
J Biol Chem. 1998 Aug 7;273(32):20007-14. doi: 10.1074/jbc.273.32.20007.
The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.
已在体外测定了胸腺嘧啶DNA糖基化酶去除胸腺嘧啶的时间进程。每个胸腺嘧啶DNA糖基化酶分子仅从含有G.T错配的DNA中去除一个胸腺嘧啶分子，因为它与去除胸腺嘧啶后留下的脱嘌呤DNA位点紧密结合。G.T错配的5'-侧翼碱基对对胸腺嘧啶的去除速率有影响：以C.G、T.A、G.C和A.T作为5'-碱基对时，催化常数（kcat）值分别为0.91、0.023、0.0046和0.0013 min-1。胸腺嘧啶DNA糖基化酶也可以从与S6-甲基硫代鸟嘌呤的错配中去除胸腺嘧啶，但与G.T错配不同，5'-C.G对速率没有显著影响：以C.G、T.A、G.C和A.T作为5'-碱基对时，从SMeG.T中去除胸腺嘧啶的kcat值分别为0.026、0.018、0.0017和0.0010 min-1。当胸腺嘧啶从具有5'-侧翼C.G对的G.T错配中去除时最快，这表明该底物的快速反应涉及酶与错配鸟嘌呤的氧6或N-1氢以及5'-侧翼C.G对之间的接触。破坏这两组接触中的任何一组（即用T.A替换5'-侧翼C.G碱基对或用SMeG.T替换G.T错配）对速率的影响与破坏两组接触（即用TpSMeG.T替换CpG.T）基本相同，因此这些接触可能是协同的。该糖基化酶从G.U、C.U和T.U碱基对中去除尿嘧啶的速度比从G.T中去除胸腺嘧啶的速度快。它甚至可以从A.U碱基对中去除尿嘧啶，尽管速率非常低。因此，胸腺嘧啶DNA糖基化酶可能对更高效的通用尿嘧啶DNA糖基化酶起到备份作用。
