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Structural basis of interstrand cross-link repair by O6-alkylguanine DNA alkyltransferase
Organic & Biomolecular Chemistry ( IF 2.9 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1039/c7ob02093g Alexey Y. Denisov 1, 2, 3, 4 , Francis P. McManus 1, 2, 3, 4 , Derek K. O'Flaherty 1, 2, 3, 4 , Anne M. Noronha 1, 2, 3, 4 , Christopher J. Wilds 1, 2, 3, 4
Organic & Biomolecular Chemistry ( IF 2.9 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1039/c7ob02093g Alexey Y. Denisov 1, 2, 3, 4 , Francis P. McManus 1, 2, 3, 4 , Derek K. O'Flaherty 1, 2, 3, 4 , Anne M. Noronha 1, 2, 3, 4 , Christopher J. Wilds 1, 2, 3, 4
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DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O6-Alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O6-atom of 2′-deoxyguanosine and the O4-atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked duplex where two O6-atoms of 2′-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O4-atoms of thymidine was found to evade repair. ICL duplexes connecting the O4-atoms of 2′-deoxyuridine by a butylene (XLUU4) or heptylene (XLUU7) linkage have been prepared to examine the influence of the C5-methyl group on AGT-mediated repair. Both XLUU4 and XLUU7 were refractory to repair by human and E. coli (OGT and Ada-C) AGTs with comparably low μM dissociation constants for 2
:1 or 4:1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG)2, XLUU7 (Y = dU) and XLGG7 (Y = dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4–O4 bond for XLUU7 whereas a Z conformation about the C6–O6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.
中文翻译：
O 6-烷基鸟嘌呤DNA烷基转移酶修复链间交联的结构基础
DNA链间交联（ICL）是生物系统中发现的最具细胞毒性的病变之一。ø 6 -Alkylguanine DNA alkyltransferases（AGTS）能够去除从烷基化损害的直径：6 2'-脱氧鸟苷的-原子和Ò 4 -原子胸苷。人的AGT（hAGT）已显示出修复链间交联双链体的能力，其中2'-脱氧鸟苷的两个O 6原子通过丁烯（XLGG4）或庚烯（XLGG7）链接进行束缚。然而，发现胸苷的O 4原子之间的类似ICL逃避了修复。连接O 4的ICL双工已经制备了通过丁烯（XLUU4）或庚烯（XLUU7）键连接的2'-脱氧尿苷原子，以研究C5-甲基对AGT介导的修复的影响。既XLUU4和XLUU7难治由人力和修理大肠杆菌（OGT和Ada-C）用相对低μM解离常数为AGTS 2：1或4 ：1个AGT / DNA的化学计量。两个庚烯连接的DNA双链体（CGAAAYTTTCG）2，XLUU7（Y = dU）和XLGG7的溶液结构（Y = dG）被求解，总体结构几乎相同，RMSD为1.22Å。发现ICL驻留在两个双工的主凹槽中。对于XLUU7，该键采用关于C4- O 4键的E构象，而对于XLGG7，则观察到关于C6- O 6键的Z构象。这种E对Z构象可能部分解释了hAGT对这些ICL修复的区分，这是由hAGT的晶体结构与各种底物一起支持的，这些底物已被观察为Z构象。此外，XLUU7在ICL站点上具有更高的移动性相对于可能在hAGT修复中发挥作用的XLGG7观察到“异位”。综上所述，这些发现就其处理能力和底物特异性而言，为AGT介导的细胞毒性ICL修复提供了见识。
更新日期：2017-09-22
:1 or 4:1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG)2, XLUU7 (Y = dU) and XLGG7 (Y = dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4–O4 bond for XLUU7 whereas a Z conformation about the C6–O6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.
中文翻译：
O 6-烷基鸟嘌呤DNA烷基转移酶修复链间交联的结构基础
DNA链间交联（ICL）是生物系统中发现的最具细胞毒性的病变之一。ø 6 -Alkylguanine DNA alkyltransferases（AGTS）能够去除从烷基化损害的直径：6 2'-脱氧鸟苷的-原子和Ò 4 -原子胸苷。人的AGT（hAGT）已显示出修复链间交联双链体的能力，其中2'-脱氧鸟苷的两个O 6原子通过丁烯（XLGG4）或庚烯（XLGG7）链接进行束缚。然而，发现胸苷的O 4原子之间的类似ICL逃避了修复。连接O 4的ICL双工已经制备了通过丁烯（XLUU4）或庚烯（XLUU7）键连接的2'-脱氧尿苷原子，以研究C5-甲基对AGT介导的修复的影响。既XLUU4和XLUU7难治由人力和修理大肠杆菌（OGT和Ada-C）用相对低μM解离常数为AGTS 2
：1或4 ：1个AGT / DNA的化学计量。两个庚烯连接的DNA双链体（CGAAAYTTTCG）2，XLUU7（Y = dU）和XLGG7的溶液结构（Y = dG）被求解，总体结构几乎相同，RMSD为1.22Å。发现ICL驻留在两个双工的主凹槽中。对于XLUU7，该键采用关于C4- O 4键的E构象，而对于XLGG7，则观察到关于C6- O 6键的Z构象。这种E对Z构象可能部分解释了hAGT对这些ICL修复的区分，这是由hAGT的晶体结构与各种底物一起支持的，这些底物已被观察为Z构象。此外，XLUU7在ICL站点上具有更高的移动性相对于可能在hAGT修复中发挥作用的XLGG7观察到“异位”。综上所述，这些发现就其处理能力和底物特异性而言，为AGT介导的细胞毒性ICL修复提供了见识。
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