LESION-BYPASS DNA POLYMERASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们目前正在研究来自Solfararicus的DinB同源(DBH)损伤旁路DNA聚合酶，并将在明年将我们的工作扩展到人类DinB同源(Pol Kappa)。在接下来的两年里，我们将重点关注这些病变旁路聚合酶的两个方面： A.未损坏DNA模板的错误特异性。当遇到特定的热点序列时，-1\f25 DBH-1聚合酶以高频率进行移码缺失。这些热点由模板链中的2或3个嘧啶组成，模板链的侧翼是鸟苷。当酶遇到这些热点时，它50%的时间会跳过其中一个模板嘧啶。这种极高的错误率可能与S对结合脱氧胞苷的酶的偏好有关(与其他3个核苷酸相比，10倍的偏好)。我们想要了解产生这些错误的结构机制，以及跳过模板嘧啶和优先将胞苷掺入引物链的序列特异性。 我们已经结晶并(通过分子置换)解决了DBH与DNA的络合结构，其中DNA在两个不同的位置含有一个跳过的模板碱基。在与活性位点相对的一个位置，碱基从引物-模板双链中凸出，在那里它只与溶剂接触，而不与蛋白质接触。在另一个位置，代表跳过碱基后两个核苷酸被结合的阶段，碱基从引物-模板双链中凸出，在那里它结合在酶上的一个小口袋中(见附图)。我们目前正在将这些结构改进到3.0和2.7的高分辨率极限。 我们在明年的目标是确定共晶体结构，其中膨胀的碱基已经改变为其他3个核苷酸中的每一个(我们目前的结构以胞苷为膨胀的碱基)。这些结构将提供有关酶跳过嘧啶模板碱基的偏好的信息。通过将凸起的碱基放置在模板链中的几个不同位置，我们希望了解-1\f25 Frame Shift-1错误首先是如何产生的，然后是如何传播的。此外，我们计划确定4个传入核苷酸中的每一个与互补模板碱基配对的结构。 B.DNA模板病变的旁路。DBH最近被证明通过在受损的模板碱基对面加入脱氧胞苷来有效地绕过脱氧鸟苷的N_2-呋喃加合物。在这项工作的第二年，我们计划通过确定DBH与含有脱氧鸟苷模板碱基的DNA与各种N-加合物的DNA络合物的共晶结构，来研究这种病变旁路的特异性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are currently studying in the DinB homolog (Dbh) lesion bypass DNA polymerase from S. solfataricus and will extend our work to the human DinB homolog (Pol kappa) within the next year. In the next two years we will focus on two aspects of these lesion-bypass polymerases: a. Error specificity on undamaged DNA templates. The Dbh polymerase makes -1 frameshift deletions at a high frequency when it encounters specific ¿¿¿¿¿¿hotspot¿¿¿¿¿¿ sequences. These hotspots consist of a run of 2 or 3 pyrimidines in the template strand flanked on the 5¿¿¿¿¿¿ side by a guanosine. When the enzyme encounters these hotspots, it skips one of the templating pyrimidines 50% of the time. This tremendously high error rate is probably linked to the enzyme¿¿¿¿¿¿s preference for incorporating deoxycytidine (10-fold preference compared to the other 3 nucleotides). We want to understand the structural mechanism by which these errors are generated and the sequence specificity for skipping template pyrimidines and for preferentially incorporating cytidine into the primer strand. We have crystallized and solved (by molecular replacement) the structures of Dbh complexed with DNA containing a skipped template base at two different positions. In one position, just opposite the active site, the base is bulged out of the primer-template duplex where it is only in contact with solvent, not protein. In the other position, representing a stage where two nucleotides have been incorporated after the base was skipped, the base is bulged out of the primer-template duplex where it is bound in a small pocket on the enzyme (see attached figure). We are currently refining these structures to high resolution limits of 3.0 and 2.7 . Our goals over the next year are to determine co-crystal structures where the bulged base has been changed to each of the other 3 nucleotides (our current structures have cytidine as the bulged base). These structures will provide information about the preference for the enzyme skipping pyrimidine template bases. By placing the bulged base at several different positions in the template strand, we hope to understand how -1 frameshift mistakes are first generated, and then propagated. Additionally, we plan to determine structures with each of the 4 incoming nucleotides pairing with a complementary templating base. b. Bypass of DNA template lesions. Dbh has recently been shown to bypass an N2-furfuryl adduct of deoxyguanosine efficiently by incorporating deoxycytidine opposite the damaged template base. In the second year of this work, we plan to investigate the specificity of the bypass of this lesion by determining co-crystal structures of Dbh complexed with DNA containing deoxyguanosine template bases with various N2-adducts.