RUI: Refractory Cleavage Sites to Elucidate Type IIS Restriction Endonuclease Mechanisms

凭借这一奖项，化学系的生命过程化学项目资助肯纳索州立大学(KSU)的Michael Van Dyke教授研究一种名为IIS限制性内切酶的酶，这种酶能在特定位置裂解脱氧核糖核酸(DNA)。限制性内切酶是生物技术中的关键工具，使研究人员能够制造特定的DNA片段。这些片段可以重组以产生新的DNA序列，也就是众所周知的重组DNA。IIS限制性内切酶，识别特定的DNA序列，然后在固定的距离上切割DNA。以前的工作报告说，这种切割发生时没有考虑任何特定的序列(序列特异性)。然而，现在已经鉴定出完全抵抗这种切割的序列。这个项目调查了为什么某些DNA序列不能被IIS类型的限制性内切酶切割，这可能会影响它们在生物技术中的使用。此外，这项研究结合了本科生和硕士研究生的混合，为他们提供核酸和相关酶的生物化学方面的培训。Van Dyke教授指导KSU以研究生院为导向的本科生和新生的测序核心设施的维护。这项研究项目的目的是从机械上确定为什么特定的DNA序列对IIS限制性内切酶FokI型无效。通过组合方法限制性内切酶保护、选择和扩增鉴定的抗切割序列被测序，并为基序激发分析鉴定抗切割基序。使用内部控制的限制性内切酶保护试验独立验证这些序列，以表征观察到的切割抗性随时间、温度和浓度的变化。然后使用Förster共振能量转移(FRET)进行停流光谱分析，以剖析FokI切割DNA过程中的各个步骤，并确定那些受已识别的抗切割序列影响的步骤。这项研究的信息可能为更好地理解IIS限制性内切酶对DNA的识别和切割提供了更好的机制，并可能适用于其他DNA切割/修饰系统。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Michael Van Dyke from Kennesaw State University (KSU) to investigate an enzymes called type IIS restriction endonucleases that cleaves deoxyribonucleic acid (DNA) in specific places. Restriction endonucleases are critical tools in biotechnology, allowing investigators to make specific fragments of DNA. The fragments can be recombined to create new DNA sequences, otherwise known as recombinant DNA. Type IIS restriction endonucleases, recognize specific DNA sequences, but then cleave DNA at fixed distances away from them. Previous work reported that this cleavage occurred without any regard to the specific sequence (sequence specificity). However, sequences have now been identified that are completely resistant to this cleavage. This project investigates why certain DNA sequences are unable to be cleaved by type IIS restriction endonucleases, which may impact their use in biotechnology. Also, this research incorporates a mix of undergraduate and Master's graduate students, providing them with training in the biochemistry of nucleic acids and associated enzymes. Professor Van Dyke mentors graduate school-oriented undergraduate students and maintenance of the nascent Sequencing Core Facility at KSU.  This research project is undertaken to mechanistically determine why specific DNA sequences are refractory to the type IIS restriction endonuclease FokI. Cleavage-resistant sequences identified through the combinatorial approach Restriction Endonuclease Protection, Selection, and Amplification are sequenced and resistant motifs identified for Motif Elicitation analysis. These sequences are independently validated using an internally controlled Restriction Endonuclease Protection Assay to characterize observed cleavage resistance as a function of time, temperature, and concentration. Stopped-flow spectroscopy studies are then performed using Förster resonance energy transfer (FRET) to dissect individual steps during the process of DNA cleavage by FokI and to determine those affected by the identified cleavage-resistant sequences. Information from this study may provide a better mechanistic understanding of DNA recognition and cleavage by IIS restriction endonucleases, with potential applicability to other DNA cleavage/modification systems.